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Midwest DNA Repair Symposium May 17-18 Wayne State University
16th Annual
Midwest DNA Repair
Symposium
May 17 and 18, 2014
Wayne State University
Spencer M. Partrich Auditorium
McGregor Memorial Conference Center
Detroit, MI
2
Midwest DNA Repair Symposium May 17-18 Wayne State University
16th Annual
Midwest DNA Repair
Symposium
Program Contents
Sponsors p.3
Keynote Speakers p.4
Scientific Program p.6
List of Abstracts p.12
Abstracts p.16
Author Index p. 73
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Midwest DNA Repair Symposium May 17-18 Wayne State University
16th Annual
Midwest DNA Repair Symposium
We are grateful to our sponsors
SPONSORS
$2,000 Level
$1,000 Level
Molecular Therapeutics unit
$500 Level
Travel Award
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Midwest DNA Repair Symposium May 17-18 Wayne State University
KEYNOTE SPEAKERS:
Joann Sweasy, Department of Therapeutic Radiology and
Department of Human Genetics, Yale University School of Medicine
Base excision repair (BER) plays a fundamental role in the
preservation of genome integrity. This pathway repairs
approximately 20,000 DNA base lesions per cell per day. DNA
polymerase beta (Pol β) is a key enzyme in the BER pathway,
that prevents accumulation of intermediate DNA repair
substrates, such as single nucleotide gaps, that lead to genomic
instability. Because mice deficient in POLB are not viable, the
function of this polymerase is not known within the context of
the whole organism. The Y265C variant of Pol β catalyzes DNA
synthesis at a significantly slower rate than wild-type Pol β
(WT). Here we show that knock-in mice expressing the Y265C
Pol β variant develop autoimmune pathology strongly resembling Systemic Lupus
Erythematosus (SLE). SLE is a multi-organ disease, characterized by the presence of
autoantibodies that form immune complexes and lead to tissue damage. As in SLE, the
POL BY265C/C mice exhibit increased levels of dermatitis, antinuclear antibody (ANA),
and renal disease compared to their WT siblings. Of note, the immunoglobulin heavy
chain junctions from the POL BY265C/C mice have shorter lengths, and somatic
hypermutation is dramatically increased. These results indicate that compromised Pol
β function during the generation of immune diversity leads to lupus-like disease in
mice, and raise the possibility that compromised DNA repair could be an underlying
cause of autoimmune disease in humans.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Thomas Glover, Department of Human Genetics and Department
of Pathology, University of Michigan
Copy number variants (CNVs), defined as deletions or
duplications of 50 bp to over a Mb, arise at a high
mutation rate and are a major factor in genomic
structural variation, numerous genetic disorders and
cancer. However, there is limited understanding of the
mechanisms by which many CNVs arise, both in terms of
initial DNA insult and aberrant repair, and the risk factors
involved. Over the past seven years, we have
demonstrated that CNVs are readily induced by agents
that lead to replication stress in cultured human cells.
Both simple and complex CNVs are induced that are
characterized by microhomologous breakpoint junctions
indicative of erroneous nonhomologous repair. They
closely resemble the “nonrecurrent” class of CNVs and
provide a model system for mechanisms involved in their formation. Genomic
hotspots for these CNVs were found, some of which coincided with common fragile
sites (CFSs). Working with large sets of de novo CNVs and metaphase breaks from
cultured cell lines, we have recently demonstrated that CNVs and CFSs are different
manifestations of replication stress at the same human loci. A combination of gene
size, active transcription and late replication acting in a cell-type specific manner
impacts sites of instability and allows predictions of instability hotspots in any cell
type. A unified model emerges in which large active transcription units drive extreme
locus instability due to a high susceptibility to fork failure, especially double fork-
failures followed by template switching or MMBIR. These findings define CNV
hotspots and CFSs as loci where a dangerous combination of large active transcription
units and late replication drives extreme local genomic instability under replication
stress.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Scientific Program
16th Annual Midwest DNA Repair Symposium
May 17-18, 2014
Scientific Sessions, Lunch, Breakfast: Spencer M Partrich Auditorium
Banquet, Posters, Reception: McGregor Memorial Conference Center
Saturday, May 17, 2014
11:00-1:00 Registration Spencer M Partrich Atrium
11:00-12:00 Lunch (boxed) Spencer M Partrich Atrium
Poster set-up McGregor Memorial Conference Center
12:00-12:15 Welcome
Dr. Wayne Raskind, Dean, College of Liberal Arts and Sciences (CLAS)
Introduction: Diane Cabelof
Keynote Speaker
12:15-1:15 Joann Sweasy (Introduction: Dr. G. Andrés Cisneros)
“DNA Repair and Autoimmunity”
Oral Symposium 1 DNA Repair and Human Disease Associations
Session Chair: Ahmad Heydari
1:15-1:30 A recurrent cancer-associated DNA polymerase epsilon mutation causes
an exceptionally strong mutator phenotype
Daniel P. Kane and Polina V. Shcherbakova
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska
Medical Center
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Midwest DNA Repair Symposium May 17-18 Wayne State University
1:30-1:45 Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells
to cisplatin
Qi-En Wang, Amit Kumar Srivastava, Chunhua Han, Ran Zhao, Tiantian Cui, Altaf
A. Wani
Department of Radiology, The Ohio State University
1:45-2:00 Folate restriction provides protection against colon carcinogenesis in DNA
polymerase β haploinsufficient mice: modulation of mTOR pathway
impacting cancer and aging
Safa Beydoun, Archana Unnikrishnan, Michael FitzGerald, Lisa Ventrella-
Lucente, Sukayna Ismail, Ali Fardous, Ahmad Heydari.
Department of Nutrition and Food Science, Wayne State University
2:00-2:15 Role of DNA polymerase β in premature senescence of Down syndrome
Aqila A. Ahmed, MieJung Park-York, Diane C. Cabelof
Department of Nutrition and Food Science, Wayne State University
2:15-2:30 Stem cell mutagenesis and carcinogenesis in the colon of wild type and
immune compromised mice after treatment with azoxymethane and/or
dextran sodium sulfate
Ryan Whetstone, Shih-Fan Kuan, Barry Gold
University of Pittsburgh
BREAK
2:30-3:00
Keynote Speaker 3:00-4:00 Dr. Thomas Glover (Introduction: Dr. Henry Heng)
“Unifying the Mechanisms of Replication Stress-Induced Fragile sites,
CNVs and Complex Chromosomal Rearrangements”
Coffee/Snack Break
4:00-4:15
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Oral Symposium 2 DSB repair/Replication Forks/Chromosomal Instability
Session Chair: Ashok Bhagwat
4:15-4:30 Chromosome fragmentation and non-homologous end joining lead to
genome chaos and cancer evolution
Joshua B Stevens, Guo Liu, Batoul Y. Abdallah, Steven D. Horne, Karen J. Ye,
Christine J. Ye, Henry H. Heng
Center for Molecular Medicine and Genetics, Department of Internal Medicine, and
Department of Pathology, Wayne State University School of Medicine
4:30-4:45 RAD54 family translocases counter genotoxic effects of RAD51
overexpression in human tumor cells
Jennifer M Mason, Kritika Dusad, Hillary Logan, Brian Budke, Megan Wu,
Jennifer Grubb, Ralph R. Weichselbaum, Philip P. Connell, Douglas K. Bishop
Department of Radiation and Cellular Oncology, Ludwig Center for Metastasis
Research, Department of Molecular Genetics and Cell Biology, University of
Chicago
4:45-5:00 Dissecting the telomeric and non-telomeric roles of human CST using a
STN1 separation of function mutant
Anukana Bhattacharjee, Jason Stewart, Mary F. Chaiken, Shih-Jui Hsu, Carolyn
Price
College of Medicine, University of Cincinnati
5:00-5:15 Exploring the role of Rad6 in repair of platinum-induced DNA lesions
Brittany Haynes, Matthew Sanders, Malathy Shekhar
Department of Oncology, Wayne State University and Karmanos Cancer Institute
5:15-5:30 Two replication fork maintenance pathways fuse inverted repeats to
rearrange chromosomes
Tae Moon Kim, Lingchuan Hu, Mi Young Son, Sung-A Kim, Cory L. Holland,
Satoshi Tateishi, Dong Hyun Kim, P. Renee Yew, Cristina Montagna, Lavinia C.
Dumitrache, Paul Hasty
Department of Molecular Medicine/Institute of Biotechnology, The Barshop
Institute of Longevity and Aging Studies, the University of Texas Health Science
Center at San Antonio
CHECK-IN
5:30-5:45 Judges for poster session pick up assignments from Diane
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Poster Session McGregor Memorial Conference Center
5:30-6:50
Banquet McGregor Memorial Conference Center
7:00
Reception McGregor Memorial Conference Center
9:00
Sunday May 18 2014
8:00-9:00 Continental Breakfast Spencer M Partrich Atrium
Oral Symposium 3 Ubiquitination/Pathway Coordination/Repair Kinetics
Session Chair: Malathy Shekhar
9:00-9:15 A mutant poisoning approach to determine if λ exonuclease trimers use a
sequential or non-sequential mechanism for processive digestion of
dsDNA substrates
Xinlei Pan, Charles Bell
The Ohio State University
9:15-9:30 Kinetic mechanism for the excision of εA by AlkA
Erin L. Miller, Patrick J. O’Brien
Biological Chemistry Department, University of Michigan
9:30-9:45 Transient kinetics and simulation revealed conformational changes
associated with human translesion DNA polymerase kappa
Linlin Zhao, Matthew G. Pence, Robert L. Eoff, Shuai Yuan, Catinca A. Fercu, F.
Peter Guengerich
Department of Chemistry, Central Michigan University; Department of
Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of
Medicine; Department of Biochemistry and Molecular Biology, University of
Arkansas for Medical Sciences
9:45-10:00 UbcH7 regulates 53BP1 stability and DSB repair
Xiangzi Han, Lei Zhang, Jinsil Chung, Amanda Tran, James W. Jacobberger, Ruth
Keri, Hannah Gilmore, Yowei Zhang
Department of Pharmacology, Department of Genetics and Genome Sciences,
School of Medicine, Case Western Reserve University
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Midwest DNA Repair Symposium May 17-18 Wayne State University
10:00-10:15 Novel insights into homologous recombination and cancer genetics: the
RAD51 paralog, RAD51C, functionally interacts with PALB2 and BRCA2
Jung-Young Park, Thiyam R. Singh, Nicolas Nassar, Fan Zhang, Marcel Freund,
Helmut Hanenberg, Amom Ruhikanta Meetei, Paul R. Andreassen
Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s
Research Foundation
Coffee/SnackBreak
10:15-10:30
Oral Symposium 4 Novel Approaches in DNA Repair/Intermediate repair products
Session Chair: G. Andrés Cisneros
10:30-10:45 Glioma-associated isocitrate dehydrogenase 1 mutation R132H inhibits
repair of DNA double strand breaks in human astrocytes
Robert Koncar, Lindsey Romick-Rosendale, Susanne Wells, Timothy Chan, El
Mustapha Rahassi
Division of Hematology Oncology, Department of Molecular and Cellular
Physiology, University of Cincinnati; Department of Experimental Hematology and
Cancer Biology, Cincinnati Children’s Hospital Medical Center; Department of
Radiation Oncology, Memorial Sloan-Kettering Cancer Center
10:45-11:00 Kinetic study of chromosomal double-strand breaks with diverse break
structures using high-resolution tecnhiques
Zhuobin Liang, Sivakumar Nallasivam, Thomas E. Wilson
Department of pathology, Department of Human Genetics, Department of
Molecular, Cellular and Developmental Biology, University of Michigan
11:00-11:15 Cellular processing of the small, excised, damage-containing DNA
oligonucleotide (sedDNA) products of nucleotide excision repair
Michael G. Kemp, Shobhan Gaddameedhi, Jun-Hyuk Choi, Jinchuan Hu, Aziz
Sancar
Department of Biochemistry & Biophysics, University of North Carolina School of
Medicine
11:15-11:30 Genome-wide splicing kinetics suggests differential rates of splicing and
turnover of introns
Jayendra Prasad, Karan Bedi, Brian Magnuson, Jerry Oomen, Artur Veloso,
Michelle Paulsen, Thomas E. Wilson, Mats Ljungman
Department of Radiation Oncology, Department of Human Genetics
Bioinformatics Program, Translational Oncology Program, University of Michigan
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Midwest DNA Repair Symposium May 17-18 Wayne State University
11:30-11:40 Break
11:40-11:45 17th Annual Midwest DNA Repair
11:45-12:15 Funding and Future Directions for DNA Repair
Introductions: Dr. Christopher States
Dr. Leroy Worth, NIH, Environmental Health Sciences
12:30 Awards Presentation
Graduate Student Poster, 1st and 2nd
Postdoctoral Poster, 1st and 2nd
Graduate Student Platform, 1st and 2nd
Postdoctoral Platform, 1st and 2nd
Young Investigator EMGS travel award (Peter Stambrook, presenting)
Thank you for your participation! Safe travels home.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
ABSTRACTS
Platform # Title
1. A recurrent cancer-associated DNA polymerase epsilon mutation causes an exceptionally
strong mutator phenotype
2. Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells to cisplatin
3. Folate restriction provides protection against colon carcinogenesis in DNA Polymerase β
Haploinsufficient mice; And modulates the mTOR pathway impacting cancer and aging.
4. Role of DNA polymerase β in premature senescence of Down syndrome
5. Stem Cell Mutagenesis and Carcinogenesis in the Colon of Wild Type and Immune
Compromised Mice after Treatment with Azoxymethane and/or Dextran Sodium Sulfate
6. Chromosome fragmentation and non-homologous end joining lead to genome chaos and
cancer evolution
7. RAD54 family translocases counter genotoxic effects of RAD51 overexpression in human
tumor cells
8. Dissecting the Telomeric and Non-telomeric Roles of Human CST Using a STN1
Separation of Function Mutant
9. Exploring the Role of Rad6 in Repair of Platinum-induced DNA Lesions
10. Two replication fork maintenance pathways fuse inverted repeats to rearrange chromosomes
11. A mutant poisoning approach to determine if λ Exonuclease trimers use a sequential or non-
sequential mechanism for processive digestion of dsDNA substrates
12. Kinetic mechanism for the excision of εA by AlkA
13. Transient kinetics and simulation revealed conformational changes associated with human
translesion DNA polymerase kappa
14. UbcH7 regulates 53BP1 stability and DSB repair
15. Novel insights into homologous recombination and cancer genetics: The RAD51 paralog,
RAD51C, functionally interacts with PALB2 and BRCA2
16. Glioma-associated Isocitrate Dehydrogenase 1 mutation R132H inhibits repair of DNA
double strand breaks in human astrocytes
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Midwest DNA Repair Symposium May 17-18 Wayne State University
17. Kinetic study of chromosomal double-strand breaks with diverse break structures using
high-resolution techniques
18. Cellular processing of the small, excised, damage-containing DNA oligonucleotide
(sedDNA) products of nucleotide excision repair
19. Genome-wide splicing kinetics suggests differential rates of splicing and turnover of introns
Poster # Title
1. Targeted inhibition of Replication Protein A increases Replication Stress in Cancer Cells
and Suppresses Tumor Growth
2. Translesion syntheses across O6-guanine-butylene-O6-guanine DNA interstrand cross-links
3. Functional Analysis of Conserved Amino Acids in DNA Ligase I
4. A Novel Mechanism of Hydroxyurea-Induced Cell Death in the Fission Yeast
Schizosaccharomycespombe
5. The melanocortin 1 receptor (MC1R) pathway protects against ROS-induced oxidative
stress in human melanocytes.
6. Stimulating and Sustaining ATR activity in G2/M checkpoint through ATM
Phosphorylation of RPA
7. Mre11-Cyclin Dependent Kinase 2 Interaction in the DNA Double-Strand Break Response
8. Contributions of Positively Charged DNA Binding Residues to Searching and Catalysis by
Human Alkyladenine DNA Glycosylase
9. Role of downstream mismatch repair proteins in the processing of cisplatin interstrand cross
links.
10. Developing Small Molecule Inhibitors Targeting Nucleotide Excision Repair Protein XPA
for Platinum based Combination Chemotherapy
11. Arsenic Inhibits DNA Mismatch Repair by Altering PCNA Function
12. Exploring global transcription elongation using BruDRB-Seq
13. Synthesis and evaluation of small molecule inhibitors of replication protein A
14. Catalytic Insights into Human DNA Ligase III
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Midwest DNA Repair Symposium May 17-18 Wayne State University
15. USP7 deubiquitinates XPC in response to ultraviolet light irradiation
16. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision
repair
17. Multiple Types of DNA Damage in Germinal Center-derived Human B cell Lymphomas
and Leukemias Expressing AID
18. Functional characterization of the N-terminal DNA-binding domain of Redβ: a unique
single-strand annealing protein
19. Improved promoter and enhancer signal detection in nascent RNA using BruUV-seq
20. Metabolomic profiling of triple negative breast cancer cells treated with Rad6 inhibitor
21. Sub-complexes of DNA Repair Proteins Assessed by Proximity Ligation
22. G-quadruplex sequences stall Klenow polymerase at the TCF3/PBX1 major break points in
t(1;19) translocations potentially leading to genomic instability.
23. Drug treatment fuels genome-mediated cancer evolution
24. Integrin alpha6beta4 promotes DNA repair-mediate transcriptional activation
25. Chronic maternal exposure to cigarette smoke induces a fetal DNA repair response without
a phase I xenobiotic metabolism response
26. Ab Initio QM/MM Calculations Show an Intersystem Crossing in the Hydrogen Abstraction
Step in Dealkylation Catalyzed by AlkB
27. DNA REPAIR ACTIVITIES OF MYCOBACTERIAL RMPs ARE DEFINED BY TWO
ALTERNATIVE DNA BINDING MODES.
28. A Homology Model of ABH1 Protein
29. Functional Consequences of SNPs in the RPA1 Gene
30. Nucleotide Excision Repair and Lung Cancer in Appalachian Kentucky
31. DESENSITIZATION OF BASAL CELL CARCINOMA TO THE ANTI-TUMORAL
EFFECT OF VITAMIN D: ROLE OF REDD1.
32. SAD-6: an SNF2-family protein involved in meiotic silencing by unpaired DNA
33. Investigating a DNA homology search process in the Model Organism Neurospora crassa
34. Low-dose ionizing radiation induces CNVs in cultured cells.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
35. Genome instability, the ultimate driver for cancer evolution
36. Small Molecule Inhibitors of ERCC1-XPF: A Novel Approach for Combination
Chemotherapy
37. Targeting the ERCC1/XPF Nuclease for Cancer Therapy
38. DNMT inhibitors sensitize breast cancer to radiation
39. Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells to cisplatin
40. Role of DNA polymerase β in premature senescence of Down syndrome
41. Stem Cell Mutagenesis and Carcinogenesis in the Colon of Wild Type and Immune
Compromised Mice after Treatment with Azoxymethane and/or Dextran Sodium Sulfate
42. RAD54 family translocases counter genotoxic effects of RAD51 overexpression in human
tumor cells
43. Dissecting the Telomeric and Non-telomeric Roles of Human CST Using a STN1
Separation of Function Mutant
44. A mutant poisoning approach to determine if λ Exonuclease trimers use a sequential or non-
sequential mechanism for processive digestion of dsDNA substrates
45. Kinetic mechanism for the excision of εA by AlkA
46. Glioma-associated Isocitrate Dehydrogenase 1 mutation R132H inhibits repair of DNA
double strand breaks in human astrocytes
47. Cellular processing of the small, excised, damage-containing DNA oligonucleotide
(sedDNA) products of nucleotide excision repair
48. Genome-wide splicing kinetics suggests differential rates of splicing and turnover of introns
49. Kinetic study of chromosomal double-strand breaks with diverse break structures using
high-resolution techniques
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #1
Title: A recurrent cancer-associated DNA polymerase epsilon mutation causes an
exceptionally strong mutator phenotype
Author: Daniel P. Kane, Polina V. Shcherbakova
Affiliations: Eppley Institute for Research in Cancer and Allied Diseases, University of
Nebraska Medical Center, Omaha, Nebraska
Abstract: Exonucleolytic proofreading and DNA mismatch repair (MMR) act in series to
maintain high-fidelity DNA replication and avoid mutagenesis. MMR defects
elevate overall mutation rate and are associated with increased cancer incidence.
Hypermutable colorectal and endometrial tumors with functional MMR were
recently reported to carry amino acid substitutions in the exonuclease domain of
DNA polymerase (pol) epsilon. This created a notion that loss of the proofreading
activity of pol epsilon is an initiating cause of some sporadic human cancers;
however, the functional consequences have not yet been assessed experimentally
for any of the cancer-associated pol epsilon mutations. We report here the in vivo
functional analysis of the P286R variant that is the most frequently observed
mutation in both colorectal and endometrial. Remarkably, despite its location in
the ExoI motif essential for exonucleolytic proofreading, the P286R variant
caused an extremely strong mutator phenotype, exceeding that of proofreading-
deficient pol epsilon mutants by two orders of magnitude. This explains the
unique recurrent appearance of the P286R mutation in tumors and argues that it
acts at some level other than loss of exonuclease activity to elevate cancer risk.
We present biochemical and genetic data that provide insight into the enigmatic
mutagenesis pathway activated by the P286R change. We further show that
heterozygosity for the variant allele causes a strong mutator effect comparable to
that of complete MMR deficiency, providing an explanation for why loss of
heterozygosity is not required for the development of pol epsilon-mutant human
tumors.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #2/ Poster #39
Title: Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells to
cisplatin
Authors: Qi-En Wang, Amit Kumar Srivastava, Chunhua Han, Ran Zhao, Tiantian Cui,
Altaf A. Wani
Affiliations: Department of Radiology, The Ohio State University.
Abstract: Ovarian cancer is the leading cause of gynecologic cancer deaths in the United
States. Cancer stem cells (CSCs) with enhanced tumorigenicity and
chemoresistance are believed to be responsible for treatment failure and tumor
relapse in ovarian cancer patients. However, it is still unclear how CSCs survive
DNA-damaging agent treatment. Here we analyzed the formation and removal of
cisplatin-induced 1,2-intrastrand crosslinks (Pt-GG) in CSCs, which were isolated
from ovarian cancer cell lines based on the dual expression of surface marker
CD44 and CD117. Surprisingly, our results did not support the previous
hypothesis that inefficient formation of DNA damage and enhanced DNA repair
capacity after cisplatin treatment are contributors to the cisplatin resistance of
ovarian CSCs. We further analyzed the status of the translesion DNA synthesis
(TLS), a DNA damage tolerance machinery than can help cells survive cisplatin
treatment, in both ovarian CSCs and their corresponding bulk cancer cells. Our
results revealed an enhanced expression of TLS polymerase η (Polη) at both
mRNA and protein levels in ovarian CSCs. In addition, we also observed an
increased level of monoubiquitylated PCNA (ub-PCNA), which is a key
regulatory element in TLS, in ovarian CSCs. These data indicate that CSCs may
have intrinsically enhanced TLS. To investigate the contribution of enhanced
TLS to the survival of CSCs upon cisplatin treatment, we analyzed the abundance
of CD44+CD117+ cells in various ovarian cancer cell lines after downregulation
of Polη and cisplatin treatment, as well as the sensitivity of ovarian CSCs to
cisplatin upon Polη knockdown. Our data demonstrated that downregulation of
Polη blocked cisplatin-induced CSC enrichment through the enhancement of
cisplatin-induced apoptosis in CSCs. Taken together, our data indicate that
ovarian CSCs may have intrinsically enhanced TLS, which facilitates CSCs to
survive cisplatin treatment, and leading to tumor relapse.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #3
Title: Folate restriction provides protection against colon carcinogenesis in DNA
Polymerase β Haploinsufficient mice; And modulates the mTOR pathway
impacting cancer and aging.
Authors: Safa Beydoun, Archana Unnikrishnan, Michael FitzGerald, Lisa Ventrella-
Lucente, Sukayna Ismail, Ali Fardous, Ahmad R. Heydari
Affiliations: Department of Nutrition and Food Science Wayne State University Detroit, Mi
48202
Abstract: Folate, an essential water soluble vitamin and a cofactor in one-carbon
metabolism has been associated with the etiology of many chronic diseases such
as cardiovascular disease, neurological degeneration, neural tube defect and
gastrointestinal cancers. Studies from our lab show that folate restriction (FR)
compromises the BER pathway by initiating repair without complete resolution
of the damage, resulting in accumulation of repair intermediates such as single
strand breaks. Interestingly, FR confers a protective phenotype in our BER-
deficient mice, with these animals showing significantly reduced development of
aberrant crypt foci in the colon of mice exposed to folate restriction. A diet
resulting in 90% reduction in serum folate levels modulated nutrient sensing
pathways, thereby impacting the etiology of cancer in mice. Mechanistically, our
data indicate that FR modulates nutrient sensing networks, specifically mTOR
Complex 1 (mTORC1). FR attenuates ATP levels; elevates NAD⁺ and the
NAD⁺/NADH ratio; and decreases the plasma branched-chain amino acids
leucine and isoleucine. As a result of altered purine biosynthesis, FR also
promotes accumulation of AICAR, a potent activator of AMPK. Consequently,
FR appears to increase AMPK phosphorylation and REDD1 expression, but
decreases IPMK expression; all these lead to attenuated mTORC1 kinase activity,
and thus reduced translation and proliferation and enhanced autophagy-a potential
mechanistic rationale for the anti-aging/cancer phenotype of FR. Furthermore, FR
appears to modulate survival in mice independent of food-level restriction. Our
aging colony shows an 86% survival rate for FR mice as compared to 60%
survival for a folate-supplemented group at 800 days of life.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #4/ Poster # 40
Title: Role of DNA polymerase β in premature senescence of Down syndrome
Authors: Aqila A Ahmed, MieJung Park-York, Diane C Cabelof
Affiliations: Department of Nutrition and Food Science, Wayne State University
Abstract: Down syndrome (DS) is a condition of intellectual disability characterized by
accelerated aging. Many premature aging disorders are causatively linked to
DNA repair pathways. However, accelerated aging in DS has not been directly
connected to a DNA repair defect in spite of evidence accumulated over decades
that a specific repair pathway, DNA base excision repair (BER) is reduced in DS.
We hypothesize that loss of BER in DS is causative for the observed accelerated
aging. To evaluate whether cells from DS donors senesce prematurely in
response to stress, DS and nonDS primary fibroblasts were exposed to 100μM
H2O2 for 5 days; there was a two-fold increase in stress-induced senescence in DS
fibroblasts. This accelerated senescence was accompanied by an increase in p16
expression (1.8 fold increase, p<0.01), demonstrating a predisposition for cells
from DS donors to senesce early. We also evaluated and verified the gene dosage
effect of Trisomy 21 on key oxidative stress genes localized to chromosome 21.
In the absence of exogenous oxidative stress (H2O2) there was a significant
upregulation of both CBS and SOD1 in DS donors when compared to nonDS
controls. When exogenous oxidative stress was added there was a significant
down regulation of CBS in DS cells. Bach1, a repressor of the oxidative stress
response and a Chr21 localized gene, suppresses senescence under normal
conditions. Inhibition of Bach1 by Chr21-localized miR 155 could block
senescence suppression. Our data in DS cells shows a reduction in Bach1 in fetal
samples. We hypothesized that a gene-dosage effect of a chromosome 21-linked
miRNA (miR-155) inhibited BER inducibility and promoted a damage-induced
senescence phenotype. To test this hypothesis, we ectopically overexpressed
miR-155 in MEFs; both transient and stable miR-155 overexpression
downregulated DNA polymerase beta (~50%, p<0.001). To confirm that this
occurred at the level of polB transactivation, the core polB promoter was
transfected into MEFs, resulting in a significant (50%) reduction in polB
promoter activity both at baseline and in response to MMS and H2O2 (p<0.05) in
the miR-155 overexpressing cells. Bach1 expression was also downregulated in
miR 155 cells thus allowing us to suggest that miR155 may play a key role in
inhibition of BER and polB and promotion of senescence.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #5/ Poster #41
Title: Stem Cell Mutagenesis and Carcinogenesis in the Colon of Wild Type and
Immune Compromised Mice after Treatment with Azoxymethane and/or Dextran
Sodium Sulfate
Authors: Ryan Whetstone, Shih-Fan Kuan, Barry Gold
Affiliations: University of Pittsburgh
Abstract: The induction of colon tumors and colonic stem cell mutations were evaluated
using an acute inflammation model of azoxymethane (AOM) and dextran sodium
sulfate (DSS) in wild type, TCRβ-/-, TCRδ-/- and TCRβ-/-TCRδ-/- C57Bl/6
mice. The incidence of colon tumors after AOM (15 mg/kg) followed by DSS
(1.5% in drinking water for 7 days) was: WT > TCRδ-/- > TCRβ-/-~TCRβ-/-
TCRδ-/- mice. Neither AOM or DSS alone resulted in tumors by 90 days. The
mutation frequency in colonic stem cells of wild type and TCRβ-/- mice was
evaluated after treatment with AOM 10 mg/kg) and/or DSS (2% in drinking
water for 7 days) using an enzymatic assay that monitors crypts fully populated
by cells with mutated (defective) glucose-6-phosphate dehydrogenase (G6PD)
activity. The mutation frequency in the untreated and DSS treated mice was < 10-
5. AOM alone yielded fully mutant crypts in both wild type and TCRβ-/- mice
with mutation frequencies of ~4x10-4 and 2x10-4, respectively. There was a
statistically significance decrease in the mutation frequency in the TCRβ-/-mice.
The combined treatment of AOM+DSS afforded fully mutated crypts in both
strains but with a lower M.F. than AOM alone that is attributed to the destruction
of AOM mutated crypts by the subsequent treatment with DSS. The
carcinogenicity results using the higher exposure to DSS (2% vs. 1.5%)
eliminated the tumor susceptibility difference between the WT and TCRβ-/-mice.
The results show that tumor incidence is related to DSS exposure, although DSS,
plays no role in stem mutagenesis.
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Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #6
Title: Chromosome fragmentation and non-homologous end joining lead to genome
chaos and cancer evolution
Author: Joshua B. Stevens, Guo Liu 1, Batoul Y. Abdallah
1, Steven D. Horne
1, Karen J.
Ye 1, Christine J. Ye
2, Henry H. Heng
1,3
Affiliations: 1 Center for Molecular Medicine and Genetics, Wayne State University School of
Medicine 2 Department of Internal Medcine, Wayne State University School of
Medicine 3 Department of Pathology
Abstract: Chromosomal alterations are primary contributors to cancer evolution, which is
underscored by the identification of unique, massive genomic alterations in the
majority of tumors by whole genome sequencing. These alterations often involve
recombination of multi-megabase portions of multiple chromosomes into a single
derivative chromosome. Although many hypotheses about the formation and
function of these genomes exist, the mechanism(s) behind them remain unclear.
For chaotic genomes to occur, megabase sized chromosomal pieces must be
produced and recombined to form. We show that in incomplete chromosome
fragmentation (a form of mitotic cell death) that occurs as a response to stress
produces fragments that are then rejoined. Double knockout of Ku70 and Ku80
inhibits non-homologous end joining (NHEJ) and reduces the frequency of
genome chaos as does inhibition of ligase IV activity by the inhibitor SCR7,
suggesting NHEJ plays a significant role in genome chaos. These chaotic
genomes (and their subtypes including chromothripsis, chromoplexy, and others)
rapidly and drastically alter the genome context. Alteration of genome context
results in increased diversity of replicate transcriptome profiles in cell
populations with elevated frequencies of genome chaos suggesting that genome
chaos contributes to cancer evolution by increasing transcriptome diversity of
cellular populations. Though often associated with suppression of cancer, cell
death and DNA repair mechanisms are paradoxically associated with genome
chaos resulting in the formation of new genome defined systems that play a
significant role in cancer evolution.
22
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #7/ Poster # 42
Title: RAD54 family translocases counter genotoxic effects of RAD51 overexpression
in human tumor cells
Authors: Jennifer M Mason 1, Kritika Dusad
1, Hillary Logan
1, Brian Budke
1, Megan Wu
1,
Jennifer Grubb1, Ralph R. Weichselbaum
1,2, Philip P. Connell
1, and Douglas K.
Bishop 1,3
Affiliations: 1Department of Radiation and Cellular Oncology,
2Ludwig Center for Metastasis
Research, 3Department of Molecular Genetics and Cell Biology, University of
Chicago, Chicago, IL
Abstract: The DNA repair protein RAD51 forms filaments on tracts of ssDNA leading to
repair via homologous recombination (HR). RAD51 can also bind directly to
undamaged dsDNA, although this activity is not associated with repair. The
RAD54 family DNA translocases specifically dissociate RAD51 from dsDNA. In
budding yeast, translocase mutants accumulate non-damage-associated RAD51
foci, resulting in genome instability and reduced viability. Using human tumor
cells, we show that simultaneous depletion of translocases RAD54L and
RAD54B and/or induction of RAD51 overexpression results in enhanced
accumulation of non-damage-associated RAD51 complexes. Furthermore, we
demonstrate depletion of RAD54 translocases results in reduced proliferation,
increased replication fork stalling, and mitotic defects. These results imply that
RAD54L and RAD54B counteract genome-destabilizing activities of RAD51 in
human tumor cells. In addition, treatment of human tumor cells with the RAD51-
stimulatory compound, RS-1, results in accumulation of non-damage associated
RAD51 complexes resulting in cell death. Finally, we demonstrate treatment with
RS-1 significantly reduces tumor size in a xenograft mouse model. Thus,
inhibition of RAD54 family translocases and/or direct stabilization of toxic
RAD51 complexes may be effective in the treatment of cancer.
23
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #8/ Poster # 43
Title: Dissecting the Telomeric and Non-telomeric Roles of Human CST Using a STN1
Separation of Function Mutant
Authors: Anukana Bhattacharjee, Jason Stewart, Mary F. Chaiken, Shih-Jui Hsu, Carolyn
Price
Affiliations: College of Medicine, University of Cincinnati
Abstract Human CST (CTC1-STN1-TEN1) is an RPA-like, ssDNA-binding complex that
plays a novel role in telomere replication. Human STN1 and TEN1 crystal
structures are strikingly similar to RPA2 and RPA3 respectively and bear OB fold
domains which are important for ssDNA binding and protein-protein interactions.
Although no crystal structures are available, structure prediction with CTC1
shows similarity with RPA1 in having multiple OB fold domains. CTC1 and
STN1 were originally identified as a DNA polymerase alpha stimulatory factor
(AAF) that enhances pol α processivity and template affinity. Our group has
shown that, similar to the budding yeast CST (Cdc13-Stn1-Ten1) complex,
human CST functions later in telomere replication after telomerase mediated
elongation of the G-rich strand, to promote fill in synthesis of the complementary
C-Strand. Human CST also facilitates the earlier replication of the telomere
duplex DNA. Unlike yeast CST, the human complex seems to have no direct role
in telomere protection. Depletion of STN1 leads to an increase in multi-telomere
signals (MTS), increased length of the telomeric G-overhang and a lag in
telomere duplex replication. CST is not only important for telomere replication,
but also plays roles in genome wide replication rescue. Depletion of STN1 leads
to an increase in anaphase bridges in the absence of telomeric fusions.
Furthermore, we found that depletion of STN1 leads to a decrease in nucleotide
(EdU) uptake following hydroxyurea (HU)-induced fork stalling. We also
observed a concomitant decrease in new origin firing. These findings indicate that
CST plays a more general role in genome-wide replication restart. Our results
suggest that human CST functions as a specialized DNA replication factor that
promotes the rescue of stalled replication within the telomere duplex region and
across the genome. To further dissect the telomeric verses genome wide roles of
CST, we created a cell line stably expressing a mutated form of STN1 with three
amino acid changes in the conserved OB domain. We and others have shown that
expression of this mutant STN1 in STN1 knockdown cells greatly reduces the
DNA binding capacity of CST. The STN1 mutant rescues the telomeric C-strand
fill in function and genome-wide replication restart defects associated with the
STN1 knockdown but increases the anaphase bridges and MTS. Thus the OB-
fold mutant demonstrates a separation of function. We postulate that the final
mechanism whereby CST rescues telomeric or non-telomeric replication
following different forms of replication stress may ultimately be the same, e.g. by
interaction with pol α. However, our data clearly indicates that the transactions
involved in the replication rescue must involve different set of protein-DNA or
protein-protein interactions.
24
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #9
Title: Exploring the Role of Rad6 in Repair of Platinum-induced DNA Lesions
Authors: Brittany Haynes, Matthew Sanders Malathy Shekhar
Affiliations: Department of Oncology, Wayne State University and Karmanos Cancer Institute
Abstract: TNBCs share several histologic features with BRCA1-related breast cancer,
which have aberrant DNA repair. Thus DNA repair pathways are thought to play
a significant role in TNBC development and therapy response. Platinum (Pt)-
based compounds induce toxic interstrand DNA crosslinks (ICLs), which require
the BRCA/Fanconi anemia (FA) and Rad6 postreplication DNA repair (PRR)
pathways for repair. Rad6 regulates PCNA ubiquitination, a critical event for
PRR, and also promotes FancD2 ubiquitination, a critical event for FA. We have
recently identified a small molecule inhibitor (SMI) of Rad6 that targets its
ubiquitin conjugating activity. We hypothesize that targeting Rad6 will be
beneficial to TNBCs treated with Pt-based compounds by preventing acquisition
of resistance and overcoming Pt resistance. MTT assays showed that MDA-MB-
231 TNBC cells have intrinsic resistance to cisplatin (IC50 12.5 μM), and
treatment with the Rad6 SMI enhances cisplatin sensitivity (IC50 reduced to 2.8
μM). Western blot analysis showed that cisplatin-induced DNA damage response
leads to PCNA and FancD2 monoubiqutitination. Treatment of MDA-MB-231
cells with the Rad6 SMI attenuated PCNA and FancD2 monoubiquitination.
Cisplatin-induced PCNA and FancD2 foci formation were diminished in cells
treated with the Rad6 SMI.We also evaluated the restart of cisplatin-induced
stalled replication forks in MDA-MB-231 cells treated with Rad6 SMI by
IdU/CldU double labeling. Our data showed that restarting of forks is observed in
vehicle and nontarget siRNA treated MDA-MB-231 cells, but rarely in cells
treated with Rad6 SMI or Rad6B siRNA. These data reveal an important role for
Rad6 in ICL repair and platinum response/resistance in TNBC.
25
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #10
Title: Two replication fork maintenance pathways fuse inverted repeats to rearrange
chromosomes
Authors: Tae Moon Kim, Lingchuan Hu, Mi Young Son, Sung-A Kim, Cory L. Holland,
Satoshi Tateishi, Dong Hyun Kim, P. Renee Yew, Cristina Montagna, Lavinia C.
Dumitrache, & Paul Hasty
Affiliations: Department of Molecular Medicine/Institute of Biotechnology, The Barshop
Institute of Longevity and Aging Studies, The University of Texas Health
Science Center at San Antonio
Abstract: Replication fork maintenance pathways maintain chromosome integrity, but their
faulty application at nonallelic repeats could generate rearrangements causing
cancer. Potential causal mechanisms are homologous recombination (HR) and
error-free postreplication repair (EF-PRR). HR repairs DNA double-strand breaks
(DSBs) and single-ended DSBs within replication. To facilitate HR, the
recombinase RAD51 and mediator BRCA2 form a filament on the DNA strand at
a break to enable annealing to the complementary sister chromatid while the
RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to
prevent recombination. HR also stabilizes and restarts replication forks without a
DSB. EF-PRR bypasses DNA incongruities that impede replication by
ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6–
RAD18 and UBC13–MMS2–RAD5 ubiquitin ligase complexes. Some
components are common to both HR and EF-PRR. Here we describe two
pathways that spontaneously fuse inverted repeats to generate unstable
chromosomal rearrangements in wild-type mouse embryonic stem (ES) cells.
Gamma-radiation induced a BLM-regulated pathway that selectively fused
identical repeats while ultraviolet light induced a RAD18-dependent pathway that
efficiently fused mismatched repeats. Moreover, TREX2 (a 3’-> 5’ exonuclease)
suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly
separating these pathways. TREX2 associated with UBC13 and enhanced PCNA
ubiquitination in response to ultraviolet light, consistent with it being a novel
member of EF-PRR. RAD18 and TREX2 also suppressed replication fork stalling
in response to nucleotide depletion. Interestingly, replication fork stalling induced
fusion for identical and mismatched repeats, implicating faulty replication as a
causal mechanism for both pathways.
26
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #11/ Poster # 44
Title: A mutant poisoning approach to determine if λ Exonuclease trimers use a
sequential or non-sequential mechanism for processive digestion of dsDNA
substrates
Authors: Xinlei Pan, Charles Bell
Affiliations: The Ohio State University
Abstract: λ Exonuclease is a highly processive 5’-3’ exonuclease that binds double-
stranded DNA ends and digests the 5’ end into mononucleotides. The digestion
product, a 3’ single-stranded overhang DNA, can serve as the substrate for
pairing proteins in homologous recombination. λ Exonuclease forms a
homotrimeric ring with a tapered central channel for tracking along the DNA.
Two mechanisms could be envisioned for enzymes that form oligomeric rings to
use their multiple active sites: the sequential mechanism, where all active sites in
the oligomer are engaged in catalysis sequentially; and the non-sequential
mechanism, where the substrate DNA locks onto one active site for multiple
rounds of catalysis. To understand how the λ Exonuclease trimer uses its three
active sites, we used a “mutant poisoning” approach, where inactive subunits are
mixed with active subunits to form hybrid trimers. In the sequential mechanism,
one inactive subunit will cause the trimer to lose all activity; whereas in the non-
sequential mechanism, the hybrid trimers remain active. A K131A mutant of λ
Exonuclease, which is completely inactive for nucleotide hydrolysis, but
maintains its DNA binding ability, was introduced. Nickel spin pull down assays,
where K131A mutant with a Histidine tag was mixed with untagged wild-type λ
Exonuclease and run on a nickel column, confirmed the formation of hybrid
trimers. The λ Exonuclease activity was measured by determining DNA digestion
rate. The results showed that the λ Exonuclease hybrid trimers still remain highly
active, suggesting that a non-sequential mechanism of active sites is used during
λ Exonuclease catalysis.
27
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #12/ Poster #45
Title: Kinetic mechanism for the excision of εA by AlkA
Authors: Erin L. Miller, Patrick J. O'Brien
Affiliations: Biological Chemistry Department, University of Michigan, Ann Arbor, MI
Abstract: A wide variety of DNA nucleobase lesions are generated when cells are exposed
to alkylating agents. Cellular repair pathways, including the base excision repair
(BER) pathway, have evolved to combat such damage. The bacterial 3-
methyladenine DNA glycosylase II (AlkA) and the human alkyladenine DNA
glycosylase (AAG) are independently evolved enzymes that initiate BER by
flipping a lesion out of the DNA duplex and hydrolyzing the N-glycosidic bond.
Both glycosylases have broad substrate ranges that include alkylated purines and
1-N6-ethenoadenine (εA). This study looks to characterize the kinetic mechanism
of AlkA catalyzed εA excision. As εA is excised by both glycosylases, direct
comparisons can thus be made between the kinetic mechanisms of AlkA and
AAG. Using in vitro kinetics experiments, we show that AlkA rapidly forms a
specific recognition complex with εA and that the hydrolysis of the N-glycosidic
bond is rate limiting. Stopped flow was used to directly monitor the DNA binding
and nucleotide flipping steps for AlkA. Binding and flipping occur very rapidly
in both the forward and reverse directions, indicating that the AlkA flipped out
recognition complex is relatively unstable. In contrast, AAG has been shown to
stabilize the flipped out εA complex. Additionally, while the rate of εA excision
is identical between the two proteins, AAG binds εA 1000-fold more tightly than
AlkA. Thus, AAG is the more efficient enzyme for εA excision. These data
expand our knowledge of base excision by two glycosylases that have identical
maximal repair rates, but use different mechanisms of substrate recognition.
28
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #13
Title: Transient kinetics and simulation revealed conformational changes associated
with human translesion DNA polymerase kappa
Authors: Linlin Zhao, Matthew G. Pence, Robert L. Eoff, Shuai Yuan, Catinca A. Fercu,
and F. Peter Guengerich
Affiliations: Department of Chemistry, Central Michigan University, Mount Pleasant,
Michigan 48859 (USA) Department of Biochemistry and Center in Molecular
Toxicology, Vanderbilt University School of Medicine, Nashville, TN, 37232
Department of Biochemistry and Molecular Biology, University of Arkansas for
Medical Sciences, Little Rock, AR, 72205 USA
Abstract: Human DNA polymerase kappa (hpol kappa) is one of the tranlesion DNA
polymerases critical for maintaining human genome integrity. To investigate the
role of conformational dynamics during hpol kappa catalysis, we created mutants
containing a single Trp residue as a fluorescence probe. A series of pre-steady
state and steady state kinetic analyses were performed using WT pol kappa and
mutants. Stopped-flow kinetic assays revealed a decrease in Trp fluorescence for
the G:dCTP pair but not for any mispairs. The decrease in fluorescence was not
rate-limiting and is considered to be related to a conformational change necessary
for correct nucleotidyl transfer. When a free 3´-hydroxyl was present on the
primer, the Trp fluorescence change returned to the baseline level at a rate similar
to the observed kcat, suggesting that this change occurs during or after
nucleotidyl transfer. However, kpol was fast, indicating that the slow
fluorescence step follows phosphodiester bond formation and is rate-limiting.
Pyrophosphate formation and release were fast and are likely to precede the
slower relaxation step. These results and simulation from global fitting of four
sets of data revealed a conformational change after the phosphodiester formation
as the rate limiting step for hpol kappa catalysis.
29
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #14
Title: UbcH7 regulates 53BP1 stability and DSB repair
Authors: Xiangzi Han1, Lei Zhang
1, Jinsil Chung
1, Amanda Tran
1, James W. Jacobberger
2,
Ruth Keri1, Hannah Gilmore
3 and Youwei Zhang
1,4
Affiliations: 1 Department of Pharmacology,
4 Department of Genetics and Genome Sciences,
School of Medicine, Case Western Reserve University, Cleveland, OH 44106,
USA
Abstract: DNA double strand break (DSB) repair is not only key to the genome stability but
is also an important anticancer target. Here we report the identification of UbcH7
(also known as Ube2L3), an ubiquitin E2 enzyme, as a novel player in DSB
repair. UbcH7 regulates ubiquitination and proteasome-dependent degradation of
53BP1. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end
resection. Therefore, UbcH7 depleted cells display increased non-homologous
end-joining (NHEJ) and reduced homologous recombination (HR) for DSB
repair. Accordingly, UbcH7 depleted cells are much more sensitive to DNA
damage than control cells likely because they mainly used the error-prone NHEJ
pathway to repair DSBs. Our studies reveal a novel layer of regulation of the
DSB repair choice and propose an innovative approach to enhance the effect of
radiotherapy or chemotherapy through stabilizing 53BP1.
30
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #15
Title: NOVEL INSIGHTS INTO HOMOLOGOUS RECOMBINATION AND
CANCER GENETICS: THE RAD51 PARALOG, RAD51C, FUNCTIONALLY
INTERACTS WITH PALB2 AND BRCA2
Authors: Jung-Young Park, Thiyam R. Singh, Nicolas Nassar,Fan Zhang, Marcel Freund,
Helmut Hanenberg, Amom Ruhikanta Meetei, Paul R. Andreassen
Affiliations: Division of Experimental Hematology and Cancer Biolgy, Cincinnati Children's
Research Foundation
Abstract: While the RAD51 paralogs, including RAD51B/C/D and XRCC2/3, are required
for DNA repair by homologous recombination (HR), little is known about their
function in this process. Adding to the uncertainty about their role, the prevailing
model is that they only form protein complexes among themselves, including
RAD51B/C/D-XRCC2 and RAD51C-XRCC3 complexes. RAD51C is of
particular interest, since RAD51C has been reported as a breast/ovarian cancer
susceptibility gene and a Fanconi anemia (FA) gene. To better understand the
function of RAD51C in HR, we have characterized RAD51C-containing protein
complexes either from untreated cells or following exposure to mitomycin C
(MMC). Surprisingly, in addition to associations with RAD51 and other RAD51
paralogs, the only other interactions found were with two components of the core
machinery for HR, PALB2 and BRCA2. Of further interest, like RAD51C,
PALB2 and BRCA2 are also associated with breast/ovarian cancer and FA.
Importantly, the C-terminal WD40 domain of PALB2 directly binds RAD51C,
XRCC3, RAD51, and BRCA2. Thus, the RAD51 paralogs, RAD51C and
XRCC3, appear to function in association with the core machinery for HR. Also,
the PALB2 WD40 domain may coordinate the function of all of these proteins in
HR. In support of this possibility, mutations of PALB2 or RAD51C found in
cancer or FA patients disrupt interactions of BRCA2, RAD51, and XRCC3 with
PALB2 or RAD51C, and compromise DNA repair. Our work yields novel insight
into mechanisms involved in HR and establishes the first system for functional
characterization of PALB2 missense mutations identified in cancer patients.
31
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #16/ Poster # 46
Title: Glioma-associated Isocitrate Dehydrogenase 1 mutation R132H inhibits repair of
DNA double strand breaks in human astrocytes
Authors: Robert Koncar1,2,
Lindsey Romick-Rosendale3, Susanne Wells
3, Timothy Chan
4,
El Mustapha Bahassi1
Affiliations: 1Division of Hematology Oncology;
2Department of Molecular and Cellular
Physiology, University of Cincinnati, Cincinnati, OH; 3Department of
Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH; 4Department of Radiation Oncology, Memorial
Sloan-Kettering Cancer Center, New York, New York
Abstract: BACKGROUND AND PURPOSE
Malignant gliomas are associated with a dismal prognosis and nearly universal
recurrence following treatment. Approximately 70-90% of grades II-III gliomas
contain a single base substitution in one Isocitrate dehydrogenase 1 (IDH1)
allele, usually at codon 132, affecting the protein’s catalytic domain. IDH1
converts isocitrate to α-ketoglutarate (αKG). However, mutant IDH1, instead,
converts αKG to 2-hydroxyglutarate. IDH1 mutation occurs early in
gliomagenesis and gliomas with the mutation respond better to treatment.
However, the role of the mutant enzyme in tumor development is unclear. We
hypothesized that mutant IDH1 impairs DNA damage repair to promote
tumorigenesis.
METHODS
Immortalized human astrocytes were transduced with an R132H mutant IDH1
gene and treated with 3Gy ionizing radiation or 20uM etoposide for 12 hours.
DNA damage was assessed by Western blot for γH2AX. Reactive oxygen species
(ROS) was measured with 2′,7′-Dichlorofluorescein diacetateusing flow
cytometry.
RESULTS
We detected higher levels of γH2AX in IDH1 mutant astrocytes compared to
controls at 3 hours (p<0.001) and 6.5 hours (p<0.05) post-irradiation and 3 hours
post-etoposide treatment, compared to IDH1 wild-type astrocytes. There was no
significant difference between IDH1 mutant and wild-type astrocyte ROS levels
at rest or following H2O2 treatment indicating a defect in DNA repair mechanisms
rather than increased ROS levels may be responsible for the genomic instability
that initiates tumor development.
CONCLUSIONS
Mutant IDH1 impairs repair of DNA double strand breaks in astrocytes.
Identifying the mechanism by which these cells accumulate damaged DNA will
help design new therapeutic strategies for treatment of malignantgliomas.
32
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #17/ Poster # 49
Title: Kinetic study of chromosomal double-strand breaks with diverse break structures
using high-resolution techniques
Authors: Zhuobin Liang, Sivakumar Nallasivam, Thomas E. Wilson
Affiliations: Department of Pathology; Department of Human Genetics; Department of
Molecular, Cellular and Developmental Biology, University of Michigan, Ann
Arbor, MI
Abstract: Non-homologous end joining (NHEJ) is the dominant double-strand break (DSB)
repair pathway in cells with limited or no 5’ resection. DSBs often harbor diverse
break structures that can complicate rejoining and lead to mutations. To better
understand how overhang polarity affects repair, we engineered an efficient
system to induce site-specific 5’-overhanging DSBs (5’ DSBs) in the S.
cerevisiae genome using zinc finger nucleases (ZFNs). Improved activity of our
ZFN system allows us to study for the first time the repair kinetics of 5’ DSBs by
chromatin immunoprecipitation and next-generation sequencing. Surprisingly,
NHEJ factors, including Yku80, Pol4 and Dnl4, had significantly higher
recruitment to ZFN-induced 5’ DSBs as compared to HO-induced 3’ DSBs in the
same locus. Consistently, NHEJ efficiency was also higher at ZFN-induced 5’
DSBs. Exonucleases involved in 5’ resection, such as Exo1, have been
demonstrated in vitro to have varying activity on substrates with different
overhang polarities. We thus hypothesize that 3’ and 5’ DSBs have different
kinetics of end-processing affecting the stability and/or activity of NHEJ. We are
in the process of analyzing the kinetics of end-processing using our newly
developed ligation-mediated qPCR at single-nucleotide resolution. In addition,
we demonstrate that yeast Tyrosyl-DNA phosphodiesterase 1 (Tdp1) was
recruited at a low level exclusively to 5’ DSBs and that its recruitment was
antagonized by Ku. Conversely, overexpression of Tdp1 weakly compromised
NHEJ. These findings suggest that Tdp1 competes with NHEJ at 5’ DSBs.
Moreover, sequencing of chromosomal 5’-DSB joints has not to date revealed
evidence for Tdp1-mediated suppression of insertional mutagenesis as observed
in plasmid studies. In summary, our study provides new insights of how overhang
polarity at genomic DSBs influences end-processing and repair outcomes.
33
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #18/ Poster # 47
Title: Cellular processing of the small, excised, damage-containing DNA
oligonucleotide (sedDNA) products of nucleotide excision repair
Authors: Michael G. Kemp, Shobhan Gaddameedhi, Jun-Hyuk Choi, Jinchuan Hu, and
Aziz Sancar
Affiliations: Department of Biochemistry & Biophysics University of North Carolina School
of Medicine Chapel Hill, North Carolina 27599
Abstract: This year marks the 50th anniversary of the discovery of nucleotide excision
repair (NER), which is a versatile system capable of removing a wide variety of
helix-distorting, bulky adducts from genomic DNA. The primary product of NER
is a ~30 nt-long oligonucleotide that contains the DNA lesion, the fate of which is
unknown. Our laboratory has recently developed methods to isolate, detect, and
characterize these small, excised, damage-containing DNA oligonucleotide
(sedDNA) products of NER in vivo in UV-irradiated human cells and mouse
tissues (Hu et al, JBC 2013; Choi et al, NAR 2014). We have found that this
methodology is applicable to a wide variety of DNA damaging agents, including
UV, benzo[a]pyrene, and cisplatin, and is capable of detecting and quantifying
excision repair events within minutes of damage induction. This sedDNA assay
should therefore be considered a new and powerful approach for studying
nucleotide excision repair in vivo. Current studies show that the primary, full-
length sedDNA products of NER remain largely associated with chromatin and in
complex with the repair/transcription factor TFIIH. Following release from
TFIIH, the sedDNAs become bound by the single-stranded DNA binding protein
RPA and undergo limited nucleolytic degradation. The inhibition of excision gap
filling activities (DNA synthesis and ligation) prevents the release of sedDNAs
from RPA and slows the rate of DNA damage removal. These findings suggest
that gap filling is tightly coordinated with sedDNA release from RPA, which
frees RPA to function in new rounds of NER and maintain genome integrity.
34
Midwest DNA Repair Symposium May 17-18 Wayne State University
Platform #19/ Poster # 48
Title: Genome-wide splicing kinetics suggests differential rates of splicing and turnover
of introns
Authors: Jayendra Prasad, Karan Bedi, Brian Magnuson, Jerry Oomen, ArturVeloso,
Michelle Paulsen, Thomas E. Wilson and Mats Ljungman
Affiliations: Department of Radation Oncology, Department of Human Genetics
Bioinformatics Program, Translational Oncology Program
Abstract: Splicing is a process wherein the spliceosome recognizes splice junctions in
nascent transcripts, excises the introns and ligates neighboring exons. Higher
eukaryotic systems have multiple introns in each gene and neither the fate of the
introns nor the kinetics with which they are spliced has been investigated on a
genome-wide scale. Here, using a new method, BruChase-seq, we demonstrate
that splicing kinetics differ between introns even when located on the same
transcript. The differences in rates was considerable and independent of gene and
intron size. Strikingly, we also found a number of spliced introns with
significantly prolonged half-lives suggesting that these introns may serve a
cellular function. The introns having these features partially overlapped across
cell lines suggesting that the functional elements (sequences) are conserved.
Collectively, we show using BruChase-seq that splicing kinetics and half-lives of
individual introns differ dramatically suggesting that these events play
physiological roles in gene regulation.
35
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #1
Title: Targeted inhibition of Replication Protein A increases Replication Stress in
Cancer Cells and Suppresses Tumor Growth
Presenting
Author:
Jason Glanzer
Other
Author(s):
Shengqin Liu, Ling Wang, Adam Mosel, Aimin Peng, Greg G. Oakley
Affiliations: University of Nebraska Medical Center Eppley Institute for Research in Cancer
Abstract: The ATR/Chk1 pathway is a critical surveillance network that maintains
genomic integrity during DNA replication by stabilizing the replication forks
during normal replication to avoid replication stress. One of the many
differences between normal cells and cancer cells is the amount of replication
stress that occurs during replication. Cancer cells with activated oncogenes
generate increased levels of replication stress. This creates an increased
dependency on the ATR/Chk1 pathway in cancer cells and opens up an
opportunity to preferentially kill cancer cells by inhibiting this pathway. In
support of this idea, we have identified a small molecule, HAMNO ((1Z)-1-[(2-
hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction
inhibitor of a protein involved in the ATR/Chk1 pathway, Replication Protein A
(RPA). HAMNO selectively binds the N-terminal domain of RPA70, effectively
inhibiting critical RPA protein interactions dependent on this domain. HAMNO
prevents etoposide-induced ATR phosphorylation of RPA32 Ser33
phosphorylation. HAMNO treatment alone induces DNA replication stress in
cancer cells that are already experiencing replication stress but not in normal
cells as detected by S-phase specific H2AX phosphorylation. HAMNO acts
synergistically with etoposide in killing cancer cells in vitro and inhibits tumor
growth in vivo. Thus, HAMNO and other protein-protein inhibitors of RPA have
potential use in cancer therapy, providing selectivity towards cancer cells by
targeting the cellular response to replication stress.
36
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #2
Title: Translesion syntheses across O6-guanine-butylene-O6-guanine DNA interstrand
cross-links
Presenting
Author:
Daniel Kool #
Other
Author(s):
Derek K. O'Flaherty § Anne M. Noronha § Christopher J. Wilds § Storm J.
Shriver # Martin Egli ¶ Linlin Zhao #
Affiliations: # Department of Chemistry, Central Michigan University, Mount Pleasant,
Michigan 48859 (USA) § Department of Chemistry and Biochemistry,
Concordia University, Montreal, Quebec H4B1R6 (Canada) ¶ Department of
Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee
37232-0146 (USA)
Abstract: DNA damage includes a variety of DNA modifications, such as DNA base
adducts, abasic sites, and intra- and interstrand cross-links. DNA interstrand
cross-links (ICLs) are two strands of DNA that are covalently linked. ICLs are
especially cytotoxic because they interfere with replication, transcription, and
recombination by preventing strand separation. One of the pathways to repair
the ICLs involves an unhooked intermediate produced by endonucleolytic
incisions adjacent to the ICL followed by translesion DNA polymerases
bypassing the lesion. Acrolein derived minor groove ICLs formed between the
exocyclic nitrogens of deoxyguanosines were previously used to test the lesion
bypass capability of translesion DNA polymerases. Due to the reactivity of the
O6-guanine atom towards various alkylating agents, O6-guanine DNA ICLs can
be potentially formed by reactions of DNA with anti-cancer drug 1,4-butanediol
dimethanesulfonate. However the miscoding potential of these O6-guanine
major groove ICLs remain elusive. We are investigating the replication
dependent ICL repair pathway for major groove DNA ICLs with O6-2′-
deoxyguanosine-butyl-O6-2′-deoxyguanosine using several lesion bypass DNA
polymerases, including Sulfolobus solfataricus DNA polymerase IV (Dpo4) and
human DNA polymerase κ. Experimental methods used include primer
extension and single base extension assays using annealed primer-template
duplexes containing a site-specifically modified O6-2′-deoxyguanosine-butyl-
O6-2′-deoxyguanosine ICL. These studies will further our understanding of
repair efficiency of major groove ICLs, which could potentially be useful for
understanding the efficacy of certain anti-cancer drugs.
37
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #3
Title: Functional Analysis of Conserved Amino Acids in DNA Ligase I
Presenting
Author:
Tom J. Jurkiw
Other
Author(s):
Mark R. Taylor and Patrick J. O'Brien
Affiliations: University of Michigan Department of Biological Chemistry
Abstract: The DNA ligase family in mammals consists of DNA ligase I, DNA ligase III
and DNA ligase IV, with DNA ligase I (LIG1) being the main ligase in the
nucleus functioning in replication and single-strand break repair. LIG1 binds to
single-stranded nicks in DNA where, in the presence of ATP and MgCl2, the
enzyme is able to catalyze the ligation of the DNA nick in a multi-step reaction.
Previous studies have helped to elucidate the kinetic mechanism of the overall
ligation reaction, but the roles of specific amino acid residues in the reaction
have not been studied in human LIG1 in detail. Studies using the Chlorella virus
DNA ligase have identified conserved residues that, when mutated, affect
catalysis, either having a universal impact or an impact on specific steps during
ligation. In order to see if these same residues have relevance in human LIG1,
we made mutations in the conserved residue E621, located in the active site, and
in conserved residues R879 and K882, located in the OB fold domain. The
active site E621 mutation, while hypothesized to affect metal coordination,
abrogated ligation efficiency, resulting in a single-turnover rate several
magnitudes of order lower than wildtype. The OB fold double mutant was
hypothesized to result in added flexibility to the domain, allowing LIG1 to
catalyze ligation at a pre-adenylylated nick, but interestingly did not appear to
differ significantly from wildtype LIG1.
38
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #4
Title: A Novel Mechanism of Hydroxyurea-Induced Cell Death in the Fission Yeast
Schizosaccharomycespombe.
Presenting
Author:
Amanpreet Singh
Other
Author(s):
Yong-jie Xu
Affiliations: Department of Biochemistry and Molecular Biology, Boonshoft School of
Medicine, Wright State University, Dayton, OH.
Abstract: Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase (RNR) that has
been used to treat various diseases such as chronic myelogenous leukemia,
sickle cell anemia, and psoriasis. It is generally believed that its cytotoxicity is
directly caused by the DNA replication stress. Recent studies in E. coli and S.
cerevisae suggest that HU may also kill the cells by generating reactive oxygen
species (ROS). However, direct evidence remains lacking in eukaryotes,
partially because the cellular effects of ROS production and replication stress
overlap. Mutants in which the two effects can be separated will be valuable in
dissecting the mechanisms of HU-induced cell death. Here, we provide evidence
for HU-induced ROS production in a fission yeast hem13 mutant. First, the HU
sensitivity can be suppressed completely in mutant cells under anaerobic
conditions or by co-incubation with antioxidants. Second, while overexpression
of the RNR small subunit can suppress the HU sensitivity of checkpoint
mutants, it fails to rescue the hem13 mutant. Third, combination of the
checkpoint mutants with the hem13 mutation has an additive effect. Fourth, the
mutant is not sensitive to other DNA damaging agents. Finally, the levels of
ROS in HU-treated cells are increased as determined by using fluorescent dye
2',7'-dichlorodihydrofluorescein diacetate. Since hemin can rescue the mutant’s
HU sensitivity, we believe that defect in heme synthesis generates a minimal
oxidative stress, which can be exacerbated by the HU-induced ROS production.
Our study may have great implications to the treatment of cancer and fungal
diseases.
39
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #5
Title: The melanocortin 1 receptor (MC1R) pathway protects against ROS-induced
oxidative stress in human melanocytes.
Presenting
Author:
Alexandra Amaro-Ortiz
Other
Author(s):
Claci Ayers John A. D’Orazio,
Affiliations: Markey Cancer Center, Graduate Center for Toxicology, Department of
Pediatrics, University of Kentucky College of Medicine Lexington, KY, USA,
Department of Agriculture, University of Kentucky College of Art and Science
Lexington KY.
Abstract: The generation of free radicals and reactive oxidative species (ROS) are thought
to be major consequences of UV exposure, leading to cellular damage and
mutagenesis. Antioxidant enzymes are critically important in the removal of
ROS in the cell. The melanocortin 1 receptor (MC1R) signaling pathway is well
known for its role against UV resistance. Moreover, MC1R activates epidermal
melanocytes cell differentiation and cell survival. Therefore, MC1R signaling
may be an exploitable target against skin carcinogenesis. We hypothesize that
MC1R signaling regulates the expression of antioxidant enzymes, particularly
manganese superoxide dismutase (MnSOD). We used the human melanoma cell
line A375, known to harbor a loss-of-function signaling mutation in MC1R, to
determine effects of cAMP stimulation on MnSOD expression. We found
accumulation of MnSOD in the mitochondria after pharmacologic induction of
cAMP with forskolin. Furthermore, addition of an oxidative agent such as
H2O2 enhanced expression of MnSOD at the protein level as early as one hour
after MC1R stimulation. Because of this timing, we hypothesize that MC1R-
mediated MnSOD increases may not involve transcriptional up-regulation but
rather MnSOD transport and/or stability. To address the mechanism, we
transfected cells with a FLAG-tagged MnSOD and performed
immunoprecipitation to determine cAMP’s effect on possible binding partners.
Our results suggest that the MC1R signaling has a protective role in the
regulation of the oxidative injury by enhancing MnSOD in human melanocytes.
Furthermore, the protective effect of MC1R signaling may be dependent on the
levels of exogenous ROS in the cells. The MC1R/cAMP signaling pathway
holds promise as a novel preventive mechanism against UV-mediated oxidative
skin injury and melanoma.
40
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #6
Title: Stimulating and Sustaining ATR activity in G2/M checkpoint through ATM
Phosphorylation of RPA
Presenting
Author:
Shengqin Liu
Other
Author(s):
Adam Mosel, Nima Mirmoghtadaei, Jason G. Glanzer and Greg G. Oakley
Affiliations: University of Nebraska Medical Center Department of Oral Biology and Eppley
Cancer Center, Omaha, NE
Abstract: Ataxia telangiectasia and Rad3 related (ATR) protein is the key regulator of
G2/M checkpoint. Both loading and activation of ATR requires Replication
Protein A (RPA). Simultaneously, RPA is phosphorylated at the N-terminus of
the RPA2 subunit by ATR/ATM in response to DNA damage. To understand
the role RPA phosphorylation plays in the DNA damage response, we generated
wild type and S4A/S8A-RPA2 phosphomutant cell lines with endogenous
RPA2 expression stably knocked down. S4A/S8A-RPA2 phosphomutant cells
showed a defective G2/M checkpoint and failed to halt mitotic entry after
etoposide treatment in G2 of the cell cycle. Stimulation and maintenance of the
ATR-Chk1 signaling pathway, but not the initial activation of the pathway is
compromised in the RPA phosphomutant expressing cells. The stimulation of
ATR activity requires the chromatin binding and interaction of TopBP1 with
ATR. The loss of RPA2 phosphorylation results in decreased TopBP1
chromatin binding. Using ATM, DNA-PK and ATR inhibitors, ATM was
identified as the kinase responsible for phosphorylation of Ser4/Ser8 of RPA2.
Inhibiting ATM in wild type cells lead to the identical phenotype exhibited by
cells expressing the RPA2 phosphomutant. Thus, ATM and ATR work together
to maintain the G2/M checkpoint through interaction and modulation of RPA.
41
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #7
Title: Mre11-Cyclin Dependent Kinase 2 Interaction in the DNA Double-Strand
Break Response
Presenting
Author:
Mary J. Morgan(1)
Other
Author(s):
Todd A. Festerling(1,2), Jeffrey Buis(1), and David O. Ferguson(1)
Affiliations: 1) Department of Pathology, University of Michigan Medical School, Ann
Arbor, MI 48109 2) Graduate Program in Toxicology, University of Michigan
School of Public Health, Ann Arbor, MI 48109
Abstract: In eukaryotes, MRN (Mre11/Rad50/NBS1) is required for resection to initiate
homologous recombination (HR). Although required, the nuclease activities of
Mre11 alone are not sufficient for resection; BRCA1 and CtIP are needed as
well. CtIP is the factor primarily responsible for cell-cycle regulation of
resection, while the major cyclin-dependent kinase (CDK) in S-phase is CDK2
bound to Cyclin A. The phosphorylation of CtIP by CDK2 allows for assembly
of the MRN-CtIP-BRCA1 resection complex. This complex provides maximum
resection capacity for HR during the S and G2 cell cycle phases. Mre11 controls
these events through a direct interaction with CDK2 which is required for CtIP
phosphorylation and BRCA1 interaction in normally dividing cells. This
observation demonstrates that MRN has important functions both in the DDR
and in regulating the normal cell cycle. The DDR has been extensively
elucidated, but CDK2’s role within the DDR remains unclear. Thus, the present
research project investigates 1) the impact of DNA damage on the Mre11-
CDK2/Cyclin A interaction, 2) whether changes in the interaction are ATM-
dependent, and 3) whether alterations in the activity of CDK2 indicate DDR
participation. To examine the effect of DNA damage on the Mre11-
CDK2/Cyclin A complex, we exposed mammalian cells to ionizing radiation
and evaluated the interaction status of endogenous Mre11 and CDK2/Cyclin A.
Our data indicate that upon induction of DNA damage, the Mre11-
CDK2/Cyclin A complex rapidly dissociates in a manner that requires ATM
kinase activity and is correlative to a reduction in CDK2 activity.
42
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #8
Title: Contributions of Positively Charged DNA Binding Residues to Searching and
Catalysis by Human Alkyladenine DNA Glycosylase
Presenting
Author:
Yaru Zhang
Other
Author(s):
Patrick J. O'Brien
Affiliations: Chemical Biology Program and Biological Chemistry Department, University
of Michigan, MI 48109
Abstract: Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair
pathway by excising alkylated or deaminated purine lesions. AAG is thought to
use facilitated diffusion to efficiently search nonspecific DNA to find rare sites
of damage. Crystal structures of AAG in complex with damaged DNA reveals a
positively charged DNA binding surface that nicely accommodates the bend of
the extrahelical recognition complex, but it is not known whether nonspecific
DNA interactions would take advantage of the same set of electrostatic
interactions. We have individually mutated the 5 arginine and 3 lysine residues
that are near to the DNA binding interface and evaluated their contributions to
both in vitro and in vivo DNA repair. The results establish that catalytic
specificity, kcat/KM, is positively correlated with processivity, suggesting that
most residues contribute to both specific binding and nonspecific binding. A
single mutant, K210M, behaves like a separation-of-function mutant with
decreased processivity and wild-type catalytic specificity, suggesting that
nonspecific DNA binding interactions may extend beyond the specific DNA
binding site. The mutants were then tested for their ability to complement the
MMS sensitivity of a mag1 yeast deletion strain, revealing a positive correlation
between catalytic specificity and cell survival. This is consistent with the model
that cell survival requires efficient capture of cytotoxic lesions. Survival of cells
with searching deficient mutant proteins could be rescued by overexpressing the
mutant proteins. Thus, it appears that chromosomal access is not restricted and
sites of damage are readily accessible to a searching protein.
43
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #9
Title: Role of downstream mismatch repair proteins in the processing of cisplatin
interstrand cross links.
Presenting
Author:
Akshada Sawant
Other
Author(s):
Anbarasi Kothandapani, Robert Sobol, Anatoly Zhitkovich, and Steve Patrick
Affiliations: University of Toledo medical center. Univerisity of Pittsburgh. Brown
University Karmanos cancer institute, Wayne state university.
Abstract: Cisplatin is a widely used chemotherapeutic agent which is used in the
treatment of a variety of human malignancies. Cisplatin causes its cytotoxic
effects via formation of DNA adducts which disrupt various cellular processes,
ultimately leading to apoptosis. The adducts formed by cisplatin are of two
types; Intrastrand adducts (~90%) and interstrand crosslinks (ICLs) (~10%).
Despite the fewer interstrand cross links formed, these cross links are
considered to be highly cytotoxic. In our previous studies, we have shown that
cisplatin ICLs are processed by base excision repair (BER) proteins and this
processing is mutagenic and leads to activation and recruitment of mismatch
repair (MMR) proteins. Mismatch repair recognition proteins are required to
maintain a cisplatin sensitive phenotype. In addition, we show that, MLH1, a
downstream MMR protein, is essential for maintaining cisplatin sensitivity.
Loss of MLH1 resulted in the development of resistance to the drug.
Furthermore, cells displayed increased ICL repair and double strand break
repair capacity in the absence of MLH1. Similar results were observed in the
case of clinically relevant MLH1 mutants lacking ATPase activity. These
results indicate that mismatch repair processing adjacent to the cisplatin ICLs,
which is dependent on the ATPase activity, is essential for maintaining cisplatin
sensitivity. Our next aim was to study the pathways that can be targeted to
increase cisplatin sensitivity in the resistant cells. Our preliminary data reveal
that ATR and ATM kinases can be potential targets important for sensitizing
BER deficient cells to cisplatin.
44
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #10
Title: Developing Small Molecule Inhibitors Targeting Nucleotide Excision Repair
Protein XPA for Platinum based Combination Chemotherapy
Presenting
Author:
Akaash Mishra
Other
Author(s):
Derek S. Woods, SilvanaDormi and John J. Turchi
Affiliations: Department of Biochemistry and Molecular Biology, Indiana University School
of Medicine, Indianapolis, IN 46202. Department of Medicine, Division of
Hematology Oncology, Indiana University School of Medicine, Indianapolis, IN
46202.
Abstract: Resistance to platinum (Pt)-based chemotherapeutic agents has been a major
limitation for successful treatment of epithelial ovarian cancers (EOC).
Enhanced DNA repair is a major contributor for Pt-resistance. Repair of Pt-
DNA adducts occurs primarily via nucleotide excision repair (NER) and
homologous recombination repair (HRR). Germ-line mutations in BRCA1/2
predispose women to hereditary ovarian cancers that are HRR deficient. In order
to exploit the concept of synthetic lethality in Pt-based combination therapies;
we have targeted the NER pathway in HRR deficient cancers, such as BRCA1
or BRCA2 null ovarian cancer. Towards this end we have recently identified
NER inhibitors targeting the DNA binding activity of the
XerodermaPigmentosum Group A (XPA) protein, a critical component of the
NER pathway. XPA binding to damaged DNA duplex is essential for DNA
damage recognition and verification in NER and has been described as the rate-
limiting step in NER-catalyzed repair. We have employed Electrophoretic
Mobility Shift Assays to identify and characterize third-generation XPA small
molecule inhibitors. The data demonstrate a 100-fold increase in potency with
IC50 values of 1µM. Analysis of the third generation inhibitors has revealed
structure-activity relationships that define the chemical and structural features
necessary for interaction with XPA and cellular permeability. Based on these in
vitro findings, we will pursue cellular cytotoxicity and sensitization to Pt
treatment in BRCA1 null and wild type ovarian cancer cell lines, which will
form the basis for in vivo xenograft studies in mouse models for EOC.
45
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #11
Title: Arsenic Inhibits DNA Mismatch Repair by Altering PCNA Function
Presenting
Author:
Janice Ortega1
Other
Author(s):
Dan Tong2, Christine Kim1, Liya Gu1, and Guo-Min Li1
Affiliations: 1Graduate Center for Toxicology and Markey Cancer Center, University of
Kentucky College of Medicine, Lexington, Kentucky 2 Wuhan University,
Wuhan, China
Abstract: Chronic exposure to arsenic is associated with the development of certain types
of cancer, including lung and skin cancers. The exact mechanism by which
arsenic induces tumorigenesis is unknown. Previous studies have shown that
arsenic enhances the expression of epidermal growth factor receptor (EGFR),
which phosphorylates the proliferating cell nuclear antigen (PCNA), an
important mismatch repair (MMR) component. We therefore hypothesize that
arsenic induces tumorigenesis through its ability to inactivate the MMR system.
We demonstrate here, that cells exposed to arsenic express high levels of EGFR
and an increased level of phosphorylated PCNA. Interestingly, nuclear extracts
treated with arsenic or derived from arsenic-treated cells are defective in MMR;
and the arsenic-induced MMR deficiency could be reversed when non-
phosphorylated recombinant PCNA was added to the reaction. These results
suggest that arsenic inhibits MMR by promoting PCNA phosphorylation
through the activation of EGFR. Our work therefore reveals a novel mechanism
by which arsenic induces carcinogenesis.
46
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #12
Title: Exploring global transcription elongation using BruDRB-Seq
Presenting
Author:
Brian Magnuson
Other
Author(s):
ArturVeloso, Killeen S. Kirkconnell, Michelle Paulsen, Thomas E. Wilson, and
Mats Ljungman
Affiliations: Department of Radiation Oncology, University of Michigan Department of
Environmental Health Science, School of Public Health, University of Michigan
Department of Human Genetics, University of Michigan Department of
Computational Medicine and Bioinformatics, University of Michigan
Department of Pathology, University of Michigan Translational Oncology
Program, University of Michigan
Abstract: Transcription of DNA to RNA proceeds in three general steps: initiation,
elongation, and termination. The elongation process can take from minutes to
hours depending on the length of a gene and associated elongation factors.
Therefore, this step plays a critical, though sometimes overlooked, role in
timing – and therefore regulation – of gene expression. The reversible
transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole
(DRB) does not inhibit transcription initiation, but rather blocks the transition
from initiation to elongation by preventing RNA polymerase II phosphorylation.
Initiated RNAPII is released upon DRB removal. By measuring nascent RNA
synthesis with Bru-Seq following a given period of recovery after DRB
treatment, we were able to calculate transcription elongation rates across the
genome. BruDRB-Seq revealed that elongation rates vary among genes
expressed in a given cell line. Elongation was measured in multiple cell lines
and about half of genes exhibited similar rates across these cell lines, falling into
the distinct categories of fast or slow, and the rest tended to exhibit variable
and/or intermediate rates. We found that a number of sequence-specific, gene
neighborhood, and epigenetic factors correlate with elongation rate, including:
exon density, distance from nearby transcription units, and histone methylation.
BruDRB-Seq is a novel approach to measure transcription elongation and we
anticipate it to be valuable in future studies of the mechanisms of transcription
regulation.
47
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #13
Title: SYNTHESIS AND EVALUATION OF SMALL MOLECULE INHIBITORS
OF REPLICATION PROTEIN A
Presenting
Author:
SilvanaDormi
Other
Author(s):
Akaash Mishra Derek Woods John J. Turchi
Affiliations: Department of Biochemistry & Molecular Biology, Indiana University School
of Medicine, Indianapolis, IN 46202 Department of Medicine, Indiana
University School of Medicine, Indianapolis, IN 46202 NERxBioSciences Inc.,
351 W. 10th St., Suite 510, Indianapolis, IN 46202
Abstract: Cisplatin and carboplatin impart their chemotherapeutic effect by forming Pt-
DNA adducts that block DNA replication and transcription, culminating in
apoptosis. Repair of those Pt-DNA adducts via nucleotide excision repair
(NER) or homologous recombination repair (HRR) can substantially reduce the
effectiveness of the Pt therapy, contributing to cellular resistance. Hence,
inhibition of these repair pathways holds the potential to sensitize resistant
cancer cells to Pt treatment. Unlike most therapies, which are focused on
enzyme-substrate interactions, our approach addresses protein-DNA disruption,
and it is based on the hypothesis that targeting the NER pathway in HRR
deficient cancers in combination with cisplatin therapy will provide increased
efficacy with minimal toxicity. Replication protein A (RPA), a single-stranded
DNA binding protein that plays a fundamental role in the NER pathway, is the
subject of the research herein presented. After having identified a small
molecule inhibitor (SMI) of RPA with promising in vitro and cellular activity,
we synthesized analogs of the lead compound and evaluated their ability to be
used in combination therapy. Structure-activity relationship (SAR) studies led
us to the selection of an optimized lead, which showed single agent activity in
A2780 epithelial ovarian cancer cells and in a xenograft lung cancer mouse
model. These data demonstrate the utility of RPA inhibition in vivo and the
potential in the development of a novel class of anticancer therapeutics that
target protein-DNA interactions.
48
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #14
Title: Catalytic Insights into Human DNA Ligase III
Presenting
Author:
Justin R. McNally
Other
Author(s):
Patrick J. O'Brien
Affiliations: Department of Biological Chemistry, University of Michigan
Abstract: The three ATP-dependent human DNA ligases, ligase I, III, and IV, are
essential for the completion of DNA replication, repair, and recombination
pathways. Human DNA ligase III (Lig3) is unique among the human DNA
ligases due to the existence of four alternative-splice isoforms. Individual
isoforms are expressed in specific tissues and organelles, making Lig3 the only
human DNA ligase localized to both the mitochondria and the nucleus. In the
mitochondria, Lig3 plays a crucial role in DNA replication and maintenance of
the genome. However, the role of Lig3 in the nucleus is less understood. It has
been postulated that Lig3 may participate in erroneous nuclear ligation events,
possibly leading to reciprocal chromosomal translocations and/or chromosomal
fusions capable of inducing tumorigenesis. Although there are obvious
structural differences between human DNA ligase I (Lig1) and the human DNA
Lig3 isoforms, the extent of their physiological differences has yet to be
established. A direct biochemical comparison of these enzymes may provide
insight as to how the Lig3 isoforms are catalytically different from Lig1 and
from one another. To conduct a thorough characterization of the Lig3 variants,
suitable reaction conditions were identified, ensuring prolonged enzymatic
stability and activity at physiological temperature and ionic strength. Under
steady-state conditions, we investigated the Mg2+, ATP and nicked duplex
DNA substrate dependencies specific to the Lig3b isoform. Subsequent work
will focus on catalytic properties of the Lig3 isozymes, focusing on the
enzymatic contributions of the Lig3 N-terminal zinc finger, as well as the Lig3a
specific binding partner XRCC1.
49
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #15
Title: USP7 deubiquitinates XPC in response to ultraviolet light irradiation
Presenting
Author:
Jinshan He1
Other
Author(s):
Qianzheng Zhu1, Nidhi Sharma1, Gulzar Wani1, Chunhua Han1, Jiang Qian1,
Kyle Pentz1, Qi-en Wang1 and Altaf A. Wani1,2, 3
Affiliations: 1Department of Radiology, 2Department of Molecular and Cellular
Biochemistry, 3James Cancer Hospital and Solove Research Institute, The Ohio
State University, Columbus, OH 43210
Abstract: Ultraviolet light (UV)-induced Xeroderma pigmentosum complementation
group C (XPC) protein ubiquitination is mediated by an E3 ubiquitin ligase
complex containing UV damaged-DNA binding protein. Here, we report that
ubiquitin specific protease 7 (USP7) deubiquitinates XPC during NER. We
have demonstrated that transiently compromising cellular USP7, by siRNA
leads to accumulation of ubiquitinated forms of XPC. However, complete USP7
disruption causes an ubiquitin-mediated XPC degradation upon cellular
irradiation. We show that USP7 interacts with XPC in vitro and in vivo.
Overexpression of wild-type USP7, but not its catalytically inactive or
interaction-defective mutants, reduces ubiquitinated forms of XPC. Importantly,
USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination
assays in vitro. We further showed that valosin-containing protein (VCP)/p97 is
required for UV-induced XPC degradation in USP7-deficient cells. VCP/p97 is
readily recruited to DNA damage sites and co-localizes with XPC. Inhibition of
VCP/p97 causes an accumulation of ubiquitinated XPC on DNA damaged
chromatin. Moreover, USP7 disruption severely impairs the repair of
cyclobutane pyrimidine dimers (CPD) and, to a lesser extent, affects the repair
of 6-4 photoproducts (6-4PP). Taken together, our findings have uncovered an
important role of USP7 in regulating NER via deubiquitinating XPC and by
preventing its VCP/p97-regulated proteolysis (This work was supported by
grants from NIH).
50
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #16
Title: Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in
nucleotide excision repair
Presenting
Author:
Chunhua Han
Other
Author(s):
Gulzar Wani, Ran Zhao, Jiang Qian, Nidhi Sharma, Jinshan He, Qianzheng
Zhu, Qi-En Wang, Altaf A. Wani
Affiliations: Department of Radiology, The Ohio State University Wexner Medical Center.
Abstract: Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair
endonuclease, which cleaves DNA strands on the 3’ side of the DNA damage
during nucleotide excision repair (NER). In addition, XPG plays a crucial role
in initiating DNA repair synthesis through recruitment of PCNA to the repair
sites. However, the fate of XPG protein subsequent to the excision of DNA
damage has remained unresolved. Here, we show that XPG is degraded through
proteasome-mediated proteolysis upon induction of bulky lesions from
exposures to UV irradiation and cisplatin. NER process is required for XPG
degradation because both UV and cisplatin treatment-induced XPG degradation
is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In
addition, the NER-related XPG degradation requires Cdt2, a component of an
E3 ubiquitin ligase, CRL4Cdt2. Micropore local UV irradiation and in situ
Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-
damage sites and interacts with XPG in the presence of PCNA. Importantly,
Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of
DNA polymerase δ and DNA repair synthesis. Collectively, our data supports
the idea of PCNA recruitment to damage sites in conjunction with XPG,
recognition of the PCNA-bound XPG by CRL4Cdt2 for specific ubiquitylation
and protein degradation. Thus, XPG removal clears the space needed at the
damage site for the subsequent recruitment of DNA pol δ and initiation of DNA
synthesis. (This work was supported by grants from NIH).
51
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #17
Title: Multiple Types of DNA Damage in Germinal Center-derived Human B cell
Lymphomas and Leukemias Expressing AID
Presenting
Author:
Sophia Shalhout
Other
Author(s):
Angela Sosin2, Alberto Martin3, Thomas Holland4, Ayad Al-Katib2, Ashok S.
Bhagwat1
Affiliations: 1Department of Chemistry, Wayne State University, Detroit, MI 48202, USA;
2Department of Internal Medicine, Wayne State University School of Medicine,
Detroit, MI 48201; 3Department of Immunology, University of Toronto,
Toronto, ON, Canada; 4Department of Immunology and Microbiology, Wayne
State University School of Medicine, Detroit, MI 48201, USA.
Abstract: An overwhelming majority of human B lymphocyte malignancies derive from
cells that have undergone the germinal center reaction and are associated with
the expression of AID, an enzyme that converts cytosines in DNA to uracil and
is required for antibody maturation through somatic hypermutation and class-
switch recombination. Several studies have shown that these lymphoma cells
contain a higher frequency of point mutations in many genes and chromosome
translocations, but no study has yet studied genomic uracils, the likely direct
consequence of AID expression in these cancers. We show here that human B
cell lymphoma and leukemia cell lines and patient samples that overexpress
AID accumulate unprecedented levels of uracils. These uracils are present at
many loci beyond the immunoglobulin genes and unexpectedly a majority of
the uracil are not in U•G mismatches. The high genomic uracil levels in these
tumors may result from an imbalance between expression of AID and UNG
genes. These cells also contain elevated levels of other types of DNA damage
including abasic sites, and single- and double-strand breaks, and have reduced
viability. B-cell lymphomas continuously accumulate DNA lesions therefore
compromising genomic integrity, due to the action of an endogenous agent, the
enzyme AID.
52
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #18
Title: Functional characterization of the N-terminal DNA-binding domain of Redβ: a
unique single-strand annealing protein
Presenting
Author:
Christopher E. Smith
Other
Author(s):
Charles E. Bell
Affiliations: The Ohio State University
Abstract: Bacteriophage λ encodes a two-component synaptase-exonuclease (Syn-Exo)
system used for generating end-to-end concatamers of λ genome before
packaging. Redα (λ exo) is a processive 5’-3’ exonuclease that digests linear
dsDNA, yielding a 3’ ssDNA overhang. Redβ is a single-strand annealing
protein (SSAP) that binds to the resulting 3’ overhang and anneals it to a
complementary ssDNA. The current model for Redβ DNA binding and
annealing describes β binding weakly to ssDNA as an oligomeric ring of 10-15
subunits, and forming a very tight complex with newly annealed duplex in the
form of a helical filament. Redβ serves as a model to study the unique DNA
repair mechanism of single-strand annealing, which is conserved in higher
eukaryotes. We have identified a protease-resistant fragment of Redβ (1-177),
which is a target for structure determination via x-ray crystallography. We
predict the N-terminal fragment forms the DNA binding domain, while a more
flexible C-terminal tail modulates interaction with the partner exonuclease. Here
we show Redβ(FL) and Redβ(177) are both able to assemble into oligomeric
structures, but their functional properties differ significantly. Using a
fluorescence-based assay, we found Redβ(FL) preferentially binds to
sequentially-added complementary oligonucleotides, while Redβ(177) binds
more tightly to ss oligonucleotides. Utilizing a Ni-affinity pulldown assay,
Redβ(177) fails to interact with λ exonuclease. Further, we found Redβ(177) is
unable to recombine a PCR product or ss oligonucleotide with a target plasmid
containing regions of homology in vivo. Our results provide insight into how
SSAP perform their DNA binding and pairing function.
53
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #19
Title: Improved promoter and enhancer signal detection in nascent RNA using
BruUV-seq
Presenting
Author:
Killeen S Kirkconnell
Other
Author(s):
Artur Veloso, Brian Magnuson, Leonardo Carmo de Andrade Lima, Michelle
T. Paulsen, Emily A. Ljungman, Karan Bedi, Jayendra Prasad, Thomas E.
Wilson and Mats Ljungman
Affiliations: University of Michigan
Abstract (250
words or less):
We recently developed Bru-seq, a method based on metabolic labeling of
nascent RNA with bromouridine to assess transcription rates genome-wide.
Here we present BruUV-seq, which utilizes UV light to introduce
transcription-blocking DNA lesions prior to labeling and deep sequencing. By
inhibiting transcription elongation but not initiation, UV light enhances
sequencing reads near promoters and enhancers. BruUV-seq, in combination
with Bru-seq, revealed unannotated transcription start site (TSS) usage,
multiple genes being transcribed from a single TSS, and the BCR-ABL1 gene
fusion in K562 leukemia cells. While the majority of expressed genes utilized
a single TSS, we were able to identify genes with 2 to 5 active TSS. A strong
correlation was observed between the BruUV-seq signals in the first 5 kb of
genes with the Bru-seq data over the length of genes, suggesting that BruUV-
seq could be used as a surrogate for estimating nascent transcription rates.
Indeed, relative changes in transcription rates measured after TNF treatment
of HF-1 fibroblasts using the BruUV-seq signal from the TSS peaks were
similar to those measured using the Bru-seq signal from the whole gene.
Furthermore, TNF induced changes in gene expression were accompanied by
changes in eRNA production. Taken together, BrUV-seq is a powerful new
approach that can obtain very detailed information on TSS utilization, relative
nascent transcription rates, and eRNA production genome-wide.
54
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #20
Title: Metabolomic profiling of triple negative breast cancer cells treated with Rad6
inhibitor
Presenting
Author:
Nadia Saadat
Other
Author(s):
Yanhua Zhang, Brittany Haynes, Smiti Gupta, Guangzhao Mao,
MalathyShekhar
Affiliations: Wayne State University
Abstract: Rad6 is a fundamental component of the postreplication DNA repair pathway,
which allows cells to complete DNA replication despite the presence of
replication-blocking lesions induced by DNA damaging drugs. Rad6 is an
ubiquitin conjugating enzyme, and this activity is essential for Rad6 function.
Normal breast tissues have low levels of Rad6 expression. However, increases
in Rad6 expression are observed in breast hyperplasias, with overexpression in
invasive and metastatic breast cancers. Rad6 silencing in metastatic breast
cancer cell lines inhibits tumor growth, reverses EMT phenotype, and tumor
progression. These data implicate the potential benefits of targeting Rad6.
Through structure-guided pharmacophore modeling and in silico analysis, we
have developed a small molecule inhibitor (SMI 9) that selectively inhibits
Rad6 ubiquitin conjugating activity. Treatment of triple negative breast cancer
cells with SMI 9 inhibits cell proliferation and migration. To overcome the
solubility issues of SMI 9, we have developed a gold nanoparticle-based
platform for delivering SMI 9. MTT assays showed that SMI 9-GNPs and free
SMI 9 have comparable, if not superior, anti-proliferative effects on MDA-MB-
231 and SUM1315 breast cancer cells. To identify potential markers of drug
response, we performed metabolomic analysis of cells treated with blank or
SMI 9-GNPs. Principle component analysis score plots showed cell-specific
differences in metabolomic profiles, and loading plots identified potential
spectral regions that were affected by drug treatment. We are in the process of
identifying the targeted metabolites.
55
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #21
Title: Sub-complexes of DNA Repair Proteins Assessed by Proximity Ligation
Presenting
Author:
Pamela VanderVere-Carozza
Other
Author(s):
John J. Turchi
Affiliations: Department of Medicine Indiana University School of Medicine, Indianapolis
IN, 46202 Department of Biochemistry and Molecular Biology, Indiana
University School of Medicine, Indianapolis IN, 46202
Abstract: Repair of damage to chromosomal DNA is orchestrated by a complex array of
proteins and their interactions. The nucleotide excision repair (NER) pathway
catalyzes the removal of bulky DNA damage including UV-photoproducts and
intrastrand DNA lesions induced by platinum-based cancer chemotherapeutics.
The non-homologous end joining (NHEJ) pathway can repair a simple DNA
double strand break by directly ligating the ends without the need for a
homologous template. Many of the interactions in these pathways have been
identified and characterized using immunoprecipitation, yeast two-hybrid
assays, co-purification and co-localization using immunofluorescence. To
interrogate a number of these interactions, we employed a proximity ligation
assay (PLA) which provides excellent resolution and quantification of specific
protein-protein associations in individual cells. The XPA-RPA interaction has
been extensively characterized and occurs independent of exogenous DNA
damage. PLA analysis confirmed this interaction using multiple antibodies to
detect the interaction in numerous human cancer cell lines. Interestingly, the
analysis of a number of other NER protein interactions was detected
independent of exogenous DNA damage. These data suggest that sub-
complexes of NER proteins exist in cells independent of exogenous DNA
damage and that assembly of an active NER complex involves pre-formed sub-
complexes. We selected the Ku70-DNA-PKcs interactions to monitor the NHEJ
pathway interactions. Again, a robust signal was detected independent of
exogenously induced DNA DSBs. Overall these results suggest that sub-
complexes of DNA repair proteins exist within cells independent of DNA
damage and may serve as a reservoir of complexes to be deployed to the sites of
damage.
56
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #22
Title: G-quadruplex sequences stall Klenow polymerase at the TCF3/PBX1 major
break points in t(1;19) translocations potentially leading to genomic instability.
Presenting
Author:
Jonathan Williams
Other
Author(s):
Erik Larson
Affiliations: Illinois State University
Abstract: Translocations are initiated by DNA breaks, which can occur upon replication
stalling, DNA damage, or other forms of genetic instability. The t(1;19)
TCF3/PBX1 translocation is responsible for 25% of all pre-B cell leukemia, and
the resulting fusion protein has also been detected in prostate and small cell
lung cancers. It is not clear why some regions of the genome are prone to DNA
breaks and translocations. However, the notion that non-B form DNA structures
contribute to genetic instability is enjoying increasing experimental support. To
further test this model we have analyzed the TCF3 and PBX1 break point
clusters for sequence motifs that can adopt G-quadruplex DNA (G4 DNA)
structures. G4 DNA is four stranded and folds from guanine repeats under
physiological conditions. This occurs when the guanine repeats are freed from
complement during transcription or replication. Using Circular Dichroism we
identified intramolecular G4 DNA structures, which map to positions 3’ and 5’
of break point clusters in TCF3 and PBX1. Using mobility shift assays,
structure formation was potassium dependent, consistent with the ionic
requirements for G4 folding. When these same sequences were positioned to
serve as the template for DNA synthesis, we observed Klenow polymerase
stalling at the guanine-rich repeats. We suggest that the break points responsible
for the t(1;19) TCF3/PBX1 translocation occur because of replication blockage
at G4 structures, implying a mechanism for genetic instability at the TCF3
locus.
57
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #23
Title: Drug treatment fuels genome-mediated cancer evolution
Presenting
Author:
Steven D. Horne
Other
Author(s):
Guo Liu, Joshua B. Stevens, Ph.D., Batoul Y. Abdallah, and Henry H.Q. Heng,
Ph.D.
Affiliations: Center for Molecular Medicine and Genetics, Wayne State University School of
Medicine, Detroit, MI
Abstract: The central paradox associated with current cancer therapeutic strategies is
initially effective treatment, which eliminates a high tumor cell count,
consistently results in successful drug resistance. Mathematical and
evolutionary modeling have previously suggested that therapeutic intervention
can provide selective pressure for the expansion of resistant variants. Drug-
related stress has been associated with genome chaos, a common phenomenon
in cancer characterized as rapid, stochastic genomic shattering and
reorganization. Since cancer represents an evolutionary process, analysis within
the context of genome-mediated cancer evolution can shed light on this key
problem of therapeutics. We propose that genomic change is a general response
to therapeutics. Drug-induced karyotypic alteration has been linked with
transcriptomic elevation, implying that drug-induced genomic change would
paradoxically provide an advantage for cancer cells through an increase of
genome heterogeneity or evolutionary potential for selection. In vivo and in
vitro models were tested using different therapeutic approaches, and surviving
cells displayed altered karyotypes for each case. To determine whether drug-
induced genome change can provide a long-term advantage to cancer cell
survival, a karyotypically stable colon cancer cell line was treated with
chemotherapy, and growth patterns were followed in a series of in vitro single-
cell and population-based experiments. Outlier treated cells displayed faster
growth rates than untreated cells, and population-based data support that these
outliers may drive cancer progression post-therapy. This macro-evolutionary
based, general mechanism of cancer drug resistance challenges the current
therapeutic aim of maximizing cancer cell death and has great implications in
the development and administration of future therapeutic strategies.
58
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #24
Title: Integrin alpha6beta4 promotes DNA repair-mediate transcriptional activation
Presenting
Author:
Brittany L. Carpenter
Other
Author(s):
Min Chen Kathleen L. O'Connor
Affiliations: University of Kentucky Markey Cancer Center Department of Molecular and
Cellular Biochemistry
Abstract: Pancreatic carcinoma has the highest death to incidence ratio of all cancers.
Integrin alpha6beta4 is overexpressed in pancreatic carcinoma, and promotes
cancer progression in part by stimulating transcriptional changes. These changes
include upregulation of ligands for the epidermal growth factor receptor,
amphiregulin and epiregulin, which are major regulators of malignancy. Based
on our previous observations that integrin alpha6beta4 alters the overall
transcriptome by targeting genes for DNA demethylation, we hypothesized that
integrin alpha6beta4 controls expression of Areg and Ereg by targeting their
respective promoters for DNA demethylation. To test this hypothesis, pancreatic
cancer cells were treated with 5-aza-2’deoxycytidine (DAC), which resulted in
increased expression of Areg and Ereg. Subsequent treatment and removal of
DAC caused stable overexpression of Areg and Ereg in low integrin
alpha6beta4-expressing cells. Similarly, treatment with the hypermethylating
agent S-adenosylmethionine decreased expression of both ligands, supporting
our hypothesis that reversible epigenetic changes are responsible for Areg and
Ereg overexpression. DNA repair has been implicated in active DNA
demethylation. To determine if DNA repair is required for Areg and Ereg
expression, pancreatic cancer cells were treated with Gemcitabine, a
chemotherapeutic that inhibits GADD45a-mediated base-excision repair (BER),
which downregulated Areg and Ereg. We also found that siRNA-mediated
knockdown or cDNA-mediated overexpression of GADD45a repressed or
elevated Areg and Ereg, respectively. Parp inhibition also suppressed
transcription of Areg and Ereg in cells with high integrin alpha6beta4. These
data indicate that integrin alpha6beta4 promotes a malignant phenotype by
regulating transcriptional expression of Ereg and Areg through DNA repair-
mediated DNA demethylation.
59
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #25
Title: Chronic maternal exposure to cigarette smoke induces a fetal DNA repair
response without a phase I xenobiotic metabolism response
Presenting
Author:
Smith, SC
Other
Author(s):
Webb C, Greene RM, Pisano MM
Affiliations: Birth Defects Center University of Louisville School of Dentistry
Abstract: Fetuses from dams exposed to cigarette smoke during gestation often exhibit
low birth weight and birth defects. Additionally, an increased likelihood of
oncogenesis looms over children exposed to cigarette smoke in utero. The
underlying mechanisms responsible for these adverse developmental outcomes
remain unclear. The constituents of cigarette smoke include polycyclic aromatic
hydrocarbons (PAHs). Phase I xenboiotic metabolism is inducible in the
developing fetus in response to PAH exposure. To determine whether chronic
maternal cigarette smoke exposure alters the xenobiotic metabolism response in
the exposed embryo, C57Bl/6 mouse dams were exposed to main- and
sidestream smoke from gestation day (gd) 1 through 17. Cigarette smoke
exposure (CSE) resulted in a significant decrease in fetal weight and length on
gd 18. Cyp1a1, canonically induced by aryl hydrocarbon receptor (AhR)
activation as part of phase I xenobiotic metabolism, was not expressed in
hepatic tissue from either the CSE or sham exposed fetuses, nor was the
expression of AhR different in CSE fetuses. PolΚ and Ercc1, however, were
induced in response to CSE, suggestive of a repair response to DNA damage.
Primary and immortalized mouse fibroblasts exposed to cigarette smoke
condensate (CSC) also induced PolΚ, and exhibited an increased micronuclei
frequency and polyploidy. Collectively, these findings suggest that the altered
fetal metabolic response to PAHs may promote an increased susceptibility to
damage-induced chromosomal aberrations.
60
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #26
Title: Ab Initio QM/MM Calculations Show an Intersystem Crossing in the Hydrogen
Abstraction Step in Dealkylation Catalyzed by AlkB
Presenting
Author:
Dong Fang
Other
Author(s):
Richard L. Lord , G. Andrés Cisneros
Affiliations: Department of Chemistry, Wayne State University
Abstract: AlkB is a bacterial enzyme that catalyzes the dealkylation of alkylated DNA
bases. The rate-limiting step is known to be the abstraction of an H atom from
the alkyl group on the damaged base by a FeIV-oxo species in the active site.
We have used hybrid ab initio quantum mechanical/molecular mechanical
methods to study this step in AlkB. Instead of forming an FeIII-oxyl radical
from FeIV-oxo near the C–H activation transition state, the reactant is found to
be an FeIII-oxyl with an intermediate-spin Fe (S = 3/2) ferromagnetically
coupled to the oxyl radical, which we explore in detail using molecular orbital
and quantum topological analyses. The minimum energy pathway remains on
the quintet surface, but there is a transition between ISFeIII-oxyl and the state
with a high-spin Fe (S = 5/2) antiferromagnetically coupled to the oxyl radical.
These findings provide clarity for the evolution of the well-known π and σ
channels on the quintet surface in the enzyme environment. Additionally, an
energy decomposition analysis reveals nine catalytically important residues for
the C–H activation step, some of which are conserved in two human
homologues. These conserved residues are proposed as targets for experimental
mutagenesis studies.
61
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #27
Title: DNA REPAIR ACTIVITIES OF MYCOBACTERIAL RMPs ARE DEFINED
BY TWO ALTERNATIVE DNA BINDING MODES.
Presenting
Author:
Mikhail Ryzhikov
Other
Author(s):
Sergey Korolev
Affiliations: Department of Biochemistry and Molecular Biology, Saint Louis University
School of Medicine St. Louis, MO
Abstract: Recombination mediator proteins (RMPs) are important for maintaining
genomic stability in all organisms after formation of DNA double-stranded
breaks (DSBs). RMPs support two distinct reactions: homologous
recombination by loading RecA-like recombinases onto ssDNA and annealing
of complementary ssDNA strands. Both reactions are inhibited by SSBs (single-
stranded DNA binding proteins) and RMPs overcome such inhibition during
DNA repair. The E. coli RecO requires interaction with SSB’s C-terminal (SSB-
Ct) to bind DNA to initiate both reactions. We previously demonstrated that
Mycobacterium smegmatis RecO does not bind SSB-Ct and its DNA binding
and annealing activities are stimulated by zinc. Here we show that
mycobacterial RecO initiates recombination independently of zinc and this
reaction is also independent of interaction with SSB-Ct. In spite of a lack of
SSB-Ct interaction, mycobacterial RecO in the presence and absence of RecR
does not displace SSB from ssDNA, suggesting that all three proteins form a
complex ssDNA. We hypothesize that the mycobacterial RecO and RecOR
complexes with SSB represent critical intermediates in annealing and
recombination. Such complexes can explain the ability of RMPs to sense stalled
replication and to support two alternative reactions in the same pathway of DSB
repair.
62
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #28
Title: A Homology Model of ABH1 Protein
Presenting
Author:
Pavel Silvestrov
Other
Author(s):
G. Andrés Cisneros*
Affiliations: Department of Chemistry, Wayne State University, Detroit, MI 48202
Abstract: ABH1 is a member of the AlkB family of proteins. These proteins have an
important role in repairing DNA from damage induced by alkylating agents. In
particular, they catalyze direct dealkylation of DNA and RNA bases.
Homologues of AlkB differ in their affinity to single or double stranded DNA
and RNA, however, the residues comprising the dealkylase active site are
conserved. The jelly-roll fold is characteristic to the structures of these
enzymes. Experimental studies have shown that ABH1 can act as a lyase at AP
sites of DNA in addition to its dealkylase function. As there is currently no
experimentally determined structure of ABH1, we carried out homology
modeling. Using homologous proteins as templates, the structure of ABH1 was
predicted in the Rosetta suite of programs. ABH1 is close to AlkB in
phylogenetic tree, and, thus, AlkB was used as a template. Duffy-binding-like
domain was also used as a template since it has high alignment scores for parts
of the sequence of ABH1. Here we present a homology model of the full ABH1
protein including the proposed AP lyase domain. MD simulations of the
predicted structures were performed. K25 is predicted to form a covalent bond
with DNA upon lyase catalysis.
63
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #29
Title: Functional Consequences of SNPs in the RPA1 Gene
Presenting
Author:
Layne Weatherford
Other
Author(s):
George Brush
Affiliations: Cancer Biology Graduate Program Department of Oncology
Abstract: Replication protein A (RPA) is a single-stranded DNA-binding protein that is
required for DNA replication, repair, and recombination. A mutation in the
large subunit of RPA (RPA1) has been shown to increase cancer risk in a mouse
model system. We will examine functional consequences of natural RPA1 SNPs
in two different systems, with attention paid to cancer-related phenotypes. In the
first system, we have created the corresponding mutations in yeast and tested
the strains for mutation frequency and sensitivity to DNA damaging agents and
a replication inhibitor. Some strains containing a variant of RFA1 (yeast
orthologue of RPA1) show slight sensitivity to ionizing radiation. Several
strains containing an RFA1 variant display a slightly higher mutation frequency
than wild type RFA1. In a preliminary study, we found some strains containing
RFA1 variants to show an increased rate of UV-induced mutagenesis. In these
cases, the mutant residue may compromise RFA1 function, at least in regard to
its role in DNA repair. For the second system, we will follow SV40 DNA
replication in vitro, a cell-free assay that relies on human DNA replication
proteins. We will use this system to determine if human SNPs affect DNA
replication efficiency. In addition, we will use this assay combined with
sequencing analysis as a novel method for determining mutation frequencies.
64
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #30
Title: Nucleotide Excision Repair and Lung Cancer in Appalachian Kentucky
Presenting
Author:
Nathaniel Holcomb
Other
Author(s):
Nathaniel Holcomb1, Mamta Goswami1, Gary Gairola1, David K. Orren1, Eric
Durbin2, Tamas S. Gal2, Isaac Hands3, Bront Davis3, Brent Hallahan4, Brent
Shelton5 Jeffrey Boyles5, Susanne Arnold6, and Isabel Mellon1
Affiliations: 1. The Graduate Center for Toxicology, College of Medicine 2. Division of
Biomedical Informatics, College of Public Health 3. Cancer Research
Informatics Shared Resource Facility, Markey Cancer Center 4. Biospecimen
Shared Resource Facility, Markey Cancer Center 5. Department of Biostatistics,
College of Public Health 6. Department of Medicine, College of Medicine
Abstract: Persistent DNA damage can result in mutations that drive carcinogenesis.
Cellular repair pathways act to remove DNA damage, minimizing genetic
changes and suppressing cancer development. Exposure to tobacco smoke
generates DNA damage causative for development of lung and other cancers;
many of the lesions are targeted specifically by the nucleotide excision repair
(NER) pathway. Thus, the efficiency of an individual’s NER system is likely to
impact cancer susceptibility, particularly in regards to tobacco smoke exposure
and lung cancer development. Although most lung cancers are related to
tobacco smoke exposure, many smokers never develop lung cancer, suggesting
additional factors involved in smoking-induced lung cancer. Chronic arsenic
exposure has been linked to lung cancer in humans. Our lab has discovered that
exposure to arsenic or cigarette smoke condensate inhibits NER efficiency in
vitro and significantly reduced the abundance of a key NER protein, XPC.
Appalachian Kentucky has an extremely high incidence of lung cancer, not
fully explained by smoking rates. Individuals in the region have elevated
exposure to trace metals (arsenic, chromium, and nickel). An epidemiological
lung cancer case-control study was initiated to collect biological and
environmental samples and personal data. We hypothesize that NER is
suppressed in individuals who develop lung cancer and we are testing this
hypothesis by measuring and comparing NER efficiency in lymphocytes
isolated from subjects in the study. We also hypothesize that exposure to trace
metals and/or tobacco smoke can inhibit the NER pathway in individuals which
in turn can lead to the development of lung cancer.
65
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #31
Title: DESENSITIZATION OF BASAL CELL CARCINOMA TO THE ANTI-
TUMORAL EFFECT OF VITAMIN D: ROLE OF REDD1.
Presenting
Author:
RawiaKhasawneh
Other
Author(s):
Dr. Ahmad Heydari Dr. ArchanaUnnikrishnan Dr. Bassel Mahmoud Dr.
IltefatHamzavi
Affiliations: Wayne State University, Henry Ford Hospital
Abstract: The relationship between Vitamin D and Non-melanoma skin Cancer is not
clear. Most of epidemiological studies are inconsistent and inconclusive, and
Mechanistic studies are lacking. Vitamin D is a seco-steroid hormone, whose
nuclear receptor (VRD) is a nuclear transcription factor. It was suggested that
vitamin D possesses anti-proliferation and pro-differentiation effect by
negatively regulating important signaling pathways. In particular, REDD1
(regulated in development and DNA damage response 1) which is a VDR target
gene inhibits mTOR pathway impacting cellular growth. In response to DNA
damage, REDD1 transcription is up-regulated by elevated levels of P53,
resulting in inhibition of mTOR pathway. Subsequently, reduction of P53
protein occurs as a feedback inhibition at the translation level. This case-control
study aimed to explore the impact of vitamin D status on the onset and
progression and possibly treatment of basal cell carcinoma. Three tissue
samples were collected from 20 BCC patients (Cancer, Proximal, and Distal),
and 6 cancer-free individuals from southeast Michigan. BCC and its feeding
cells seemed to up-regulate vitamin D activation enzymes, VDR, and its co-
activators proposing higher local activity of vitamin D. Despite the significantly
increased protein levels of REDD1 in the cancer tissue, our data showed that
VRD’s ability to down-regulate mTOR pathway through REDD1 was
diminished. Interestingly, Vitamin D negative regulation of Hedgehog-Gli
pathways was also lost in the cancer tissue. Kras mutation was detected in all
specimens obtained from cases participants. In conclusion, we propose that the
anticipated role of vitamin D is not conserved in BCC tissue.
66
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #32
Title: SAD-6: an SNF2-family protein involved in meiotic silencing by unpaired
DNA
Presenting
Author:
Dilini A. Samarajeewa
Other
Author(s):
Nick A. Rhoades, Hua Xiao, Patrick K. T. Shiu, Kevin A. Edwards, and
Thomas M. Hammond
Affiliations: School of Biological Sciences, Illinois State University, Normal, Illinois,
61790. Division of Biological Sciences, University of Missouri, Columbia,
Missouri, 65211.
Abstract: Meiotic silencing by unpaired DNA (MSUD) is a process that detects and
silences unpaired DNA between homologous chromosomes for the duration of
meiosis. It is believed that gene silencing works through an RNA-interference-
related pathway via the production of a theoretical aberrant RNA molecule.
However, the nature of the unpaired DNA scanning mechanism and the proteins
involved in the nuclear aspects of MSUD are completely unknown. Rad54 is a
protein involved in repairing double stranded DNA breaks by homologous
recombination in yeast. SAD-6 is a putative SNF2-family protein closely related
to Rad54 in Neurospora crassa. We found that MSUD is significantly
suppressed by the deletion of sad-6 locus, suggesting that it is required for the
process. Moreover, confocal microscopy studies have confirmed that GFP
tagged SAD-6 protein is localized in the nucleus during meiosis suggesting that
SAD-6 could be a protein involved in the unpaired DNA detection process of
MSUD. Further studies will seek to identify the theoretical aberrant RNA and
determine if SAD-6 is required for its production.
67
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #33
Title: Investigating a DNA homology search process in the Model Organism
Neurospora crassa
Presenting
Author:
Pegan Sauls
Other
Author(s):
Zach Smith, Kevin J. Sharp, and Thomas M. Hammond
Affiliations: Illinois State University
Abstract: When Neurospora crassa’s cells undergo meiosis, its genome undergoes a
quality control process called Meiotic Silencing by Unpaired DNA (MUSD).
MSUD scans homologous chromosomes for segments of DNA that are
unpaired. This can occur when an ectopic copy of a gene is added to a
chromosome. Since an ectopic gene copy is unpaired, it will be detected by
MSUD and silenced for the duration of meiosis. While many of the protein
players of the silencing process have been identified, those involved in the
scanning process are unknown. In our research, we have taken a genetic marker
and integrated it into different locations of the N.crassa genome. We have
discovered that unpaired genes are not always recognized by MSUD.
Essentially, recognition appears to be dependent on the distance between the
unpaired genes and their homologs, with small distances leading to unpaired
genes escaping detection. Our current work seeks to identify and characterize
the proteins involved in the detection of unpaired genes, which should help
explain the imprecision in the unpaired DNA identification process.
68
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #34
Title: Low-dose ionizing radiation induces CNVs in cultured cells.
Presenting
Author:
Martin F. Arlt
Other
Author(s):
Sountharia Rajendran, Shanda R. Birkeland, Thomas E. Wilson, Thomas W.
Glover
Affiliations: Department of Human Genetics, University of Michigan; Department of
Pathology, University of Michigan
Abstract: Copy number variants (CNVs) are deletions and duplications that can exceed 1
Mb in size. Despite their importance to human genetic variationand disease,
little is known about the molecularmechanisms and environmental risk
factorsthat impact CNV formation.While it is clear that replication stress
canlead to de novo CNVs, for example, followingtreatment of cultured
mammalian cells with aphidicolin(APH) and hydroxyurea (HU), the effectof
different types of mutagens on CNV inductionis unknown. We have
investigated the effects of ionizing radiation (IR) on CNV formation in normal
human fibroblasts. Wefound that IR in the range of 1.5–3.0 Gy
effectivelyinduces de novo CNV mutations in these cells. The IR-induced
CNVsare found throughout the genome, with the samehotspot regions seen after
APH- and HU-inducedreplication stress. IR produces duplications at ahigher
frequency relative to deletions than doAPH and HU. At most hotspots, these
duplicationsare physically shifted from the regions typicallydeleted after APH
or HU, suggestingdifferent pathways involved in their formation.CNV
breakpoint junctions from irradiated samplesare characterized by
microhomology, bluntends, and insertions like those seen in spontaneousand
APH/HU-induced CNVs and most nonrecurrentCNVs in vivo. The similarity to
APH/HU-induced CNVs suggests that low-dose IR inducesCNVs through a
replication-dependent mechanism,as opposed to replication-independentrepair
of DSBs. Consistent with this mechanism, fewer CNVs were induced by IR
when cellswere held for 48 hr before replating after irradiation.These results
predict that any environmentalDNA damaging agent that impairs replicationis
capable of creating CNVs.
69
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #35
Title: Genome instability, the ultimate driver for cancer evolution
Presenting
Author:
Henry H Heng
Other
Author(s):
Joshua Stevens, Batoul Abdallah, Steven Horne, Karen Ye, Guo Liu
Affiliations: Center for Molecular Medicine and Genetics, and Department of Pathology,
Wayne State University School of Medicine, Detroit, MI.
Abstract: The highly complex diverse genomic profile of cancer challenges current gene
mutation/pathways centered cancer research. Each specific gene mutation only
has limited predictive ability due to such a high level of complexity in each
tumor. Failure to find highly penetrant mutational drivers of cancer necessitates
searching for common drivers of cancer evolution, specifically by focusing on
the genomic organization instead of cataloging gene mutations. The genome,
rather than genes, is the platform of macro-cellular evolution meaning that
genome diversity is a pre-condition for cancer evolution. Genomic instability,
the engine of genome diversity, is the key to understanding and control cancer.
Here we discuss multiple aspects of genome instability including its
definition(s), measures, its relationship(s) with DNA repair pathways, and its
ultimate importance for cancer evolution. We then apply the genome theory
(where the karyotype rather than individual genes defines the system
inheritance) and evolutionary theory (with interaction between both punctuated
and stepwise phases) to propose a general mechanism that uses genome
instability to unify the diverse molecular causes of cancer. References: 1).
Genome chaos: survival strategy during crisis. Cell Cycle. 13:528-37 (2014). 2).
Unstable genomes elevate transcriptome dynamics. Int J Cancer. 134:2074-87
(2014). 3). Single cell heterogeneity: why unstable genomes are incompatible
with average profiles. Cell Cycle. 12:3640-9 (2013). 4). Chromosomal
instability (CIN): what it is and why it is crucial to cancer evolution. Cancer
Metastasis Rev. 32:325-40 (2013). 5). Evolutionary mechanisms and diversity
in cancer. Adv Cancer Res. 112:217-53 (2011).
70
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #36
Title: Small Molecule Inhibitors of ERCC1-XPF: A Novel Approach for Combination
Chemotherapy
Presenting
Author:
Steve M. Patrick
Other
Author(s):
Sanjeevani Arora, Vivian Kalman-Maltese, Jeffrey Sarver, Paul Erhardt and Q.
Ping Dou
Affiliations: Wayne State University/Karmanos Cancer Institute University of Toledo Health
Science Campus
Abstract: We have established a high throughput screen to identify inhibitors of the
ERCC1-XPF endonuclease. ERCC1-XPF is a critical component of the DNA
repair pathways that remove bulky DNA lesions and interstrand crosslinks from
the genome. In this role, ERCC1-XPF has a significant impact on the efficacy of
DNA damaging chemotherapeutic drugs and has been shown to affect drug
resistance. Recent literature has suggested that ERCC1 is a prognostic indicator
of chemotherapy response in lung cancer, ERCC1 deficient cells synergize with
PARP inhibitors and ERCC1 deficient cells synergize with proteins in the ATR
signaling pathway to yield a synthetic lethal interaction. These data highlight
the significance of targeting ERCC1-XPF endonuclease with small molecules to
provide novel combination therapies to achieve better patient outcomes in a
variety of cancers. In our initial screen of the NCI diversity set, we identified 2
Hits that had IC50 values in the low nanomolar range against ERCC1-XPF
while having minimal effect against two non-related endonucleases (XPG and
HhaI). In the 10-15 micromolar range, these Hits were shown to inhibit DNA
repair in cell culture and potentiate cisplatin efficacy in colony survival assays.
A structural similarity search to Hit 1 identified a natural compound from green
tea, (-)-epigallocatechin-3-gallate (EGCG), that had nanomolar potency (IC50:
50-100 nM) in the cell free assay and low micromolar (5-10 uM) activity in cell
culture. Our future studies will focus on screening structural analogs of EGCG
that are more potent ERCC1-XPF inhibitors that potentiate cisplatin effects in
ovarian and lung cancer xenografts.
71
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #37
Title: Targeting the ERCC1/XPF Nuclease for Cancer Therapy
Presenting
Author:
Derek Woods
Other
Author(s):
Shehnaz Khan, Vivian Kalman-Maltese, Kate Marchal, Steve M. Patrick, John
J. Turchi
Affiliations: NERx Biosciences, Inc.
Abstract: Cisplatin is one of the most widely prescribed chemotherapeutic agents used to
treat cancer. Cisplatin functions by damaging DNA and the effectiveness of
cisplatin in killing cancer cells is often limited by the repair of cisplatin-DNA
damage by the nucleotide excision repair (NER) and the homologous
recombination repair (HRR) pathways. The ERCC1/XPF structure-specific
nuclease functions in both of these pathways and is responsible for incision of
damaged DNA 5’ of the lesion. Expression of both ERCC1 and XPF has been
correlated with cisplatin response in ovarian and lung cancers. For this reason,
the inhibition of ERCC1/XPF nuclease activity holds the potential to enhance
cisplatin efficacy in non-responding patients that have high ERCC1 expression.
In pursuit of novel anti-cancer agents, we completed an in vitro high throughput
screen of drug like molecules for inhibitors of ERCC1/XPF. Hits were validated
in a second, gel-based assay and inhibition was confirmed to have a titratable
effect with IC50 values in the low nanomolar range. Hits were tested for
specificity of ERCC1/XPF inhibition against XPG and the HhaI restriction
enzyme. As expected, specificity varied among the validated hits. Hits were also
show to sensitize the A2780 cell line to cisplatin consistent with predicted
ERCC1/XPF inhibition. Together these results support the pursuit of
ERCC1/XPF inhibitors. Future studies will focus on determining the method of
action for these inhibitors and in vivo xenograft studies to test efficacy of leads
as adjuvant agents.
72
Midwest DNA Repair Symposium May 17-18 Wayne State University
POSTER #38
Title: DNMT inhibitors sensitize breast cancer to radiation
Presenting
Author:
Steven Zielske, Ph.D.
Other
Author(s):
Deborah A. Antwih, Kristina M. Gabbara
Affiliations: Department of Radiation Oncology, Wayne State University and Karmanos
Cancer Institute, Detroit, Michigan
Abstract: Epigenetic regulation has been shown to play a key role in the development of
many malignancies. One of the major obstacles in treating many tumor
malignancies is radiation resistance. DNA methyltransferase (DNMT) inhibitors
can restore gene expression silenced by DNA methylation. We investigated the
effects of DNMT inhibitors on radiation sensitivity in breast cancer cells. Two
breast cancer cell lines were incubated with or without 5-azacytidine for two
days before treatment with ionizing radiation and subsequently plated for
colony formation. The survival fraction of cells treated with radiation and 5-
azacytidine was lower, showing a radiation enhancement factor of 1.8 compared
to those treated with ionizing radiation alone. We further evaluated additional
DNMT inhibitors. To determine the effects of DNMT inhibitors on DNMT
levels, cells were treated with 5-azacytidine, decitabine, and zebularine. At the
end of treatment, cells were collected and lysed for Western blotting analysis to
evaluate the level of DNMT1, 3a and 3b. Pretreatment of cells with DNMT
inhibitors resulted in decreased DNMT1, and 3a, while there was no change in
DNMT3b. Each inhibitor had differential ability to reduce DNMT levels
depending on cell line. 5-Methylcytosine (5-mC) levels were quantified by
ELISA and found to be progressively reduced over time in DNMT inhibitor-
treated cells. Our results show reduced levels of 5-mC in cells treated with
DNMT inhibitors and strong radiosensitization. These data suggest that there is
a synergistic effect caused by DNMT inhibitors and ionizing radiation in the
treatment of breast cancer cells, thereby enhancing radiation toxicity.
73
Midwest DNA Repair Symposium May 17-18 Wayne State University
AUTHOR INDEX
Abdallah, Batoul Y (8) Ahmed, Aqila A (7)
Al-Katib, Ayad (51) Amaro-Ortiz, Alexandra (39)
Andreassen, Paul R (10, 30) Antwih, Deborah A (72)
Arlt, Martin F (68) Arnold, Susanne (64)
Arora, Sanjeevani (70) Ayers, Claci (39)
Bedi, Karan (10, 34, 53) Bell, Charles E (9, 26, 52)
Beydoun, Safa (7, 18) Bhagwat, Ashok S (8, 51)
Bhattacharjee, Anukana (8, 23) Birkeland, Shanda R (68)
Bishop, Douglas K (8, 22) Boyles, Jeffrey (64)
Brush, George (63) Budke, Brian (8, 22)
Buis, Jeffrey (41) Cabelof, Diane C (6, 7, 19)
Carmo de Andrade Lima, Leonardo (53) Carpenter, Brittany L (58)
Chaiken, Mary F (8, 23) Chan, Timothy (10, 31)
Chen, Min (58) Choi, Jun-Hyuk (10, 33)
Chung, Jinsil (9, 29) Cisneros, G. Andrés (6, 10, 60, 62)
Connell, Philip P (8, 22) Cui, Tiantian (7, 17)
D’Orazio, John A (39) Davis, Bront (64)
Dormi, Silvana (44, 47) Dou, Q. Ping (70)
Dumitrache, Lavinia C (8, 25) Durbin, Eric (64)
Dusad, Kritika (8, 22) Edwards, Kevin A (66)
Egli, Martin (36) Eoff, Robert L (9, 28)
Erhard, Paul (70) Fang, Dong (60)
Fardous, Ali (7, 18) Fercu, Catinca A (9, 28)
Ferguson, David O (41) Festerling, Todd A (41)
FitzGerald, Michael (7, 18) Freund, Marcel (10, 30)
Gabbara, Kristina M (72) Gaddameedhi, Shobhan (10, 33)
Gairola, Gary (64) Gal, Tamas S (64)
Gilmore, Hannah (9, 29) Glanzer, Jason G (35, 40)
Glover, Thomas W (5, 7, 68) Gold, Barry (7, 20)
Goswami, Mamta (64) Greene, RM (59)
Grubb, Jennifer (8, 22) Gu, Liya (45)
Guengerich, F. Peter (9, 28) Gupta, Smiti (54)
Hallahan, Brent (64) Hammond, Thomas M (66, 67)
Hamzavi, Iltefat (65) Han, Chunhua (7, 17, 49, 50)
Han, Xiangzi (9, 29) Hands, Isaac (64)
Hanenberg, Helmut (10, 30) Hasty, Paul (8, 25)
Haynes, Brittany (8, 24, 54) He, Jinshan (49, 50)
Heng, Henry H (7, 8, 21, 57, 69) Heydari, Ahmad (6, 7, 18, 65)
Holcomb, Nathaniel (64) Holland, Cory L (8, 25)
Holland, Thomas (51) Horne, Steven D (8, 21, 57, 69)
Hsu, Shih-Jui (8, 23) Hu, Jinchuan (10, 33)
Hu, Lingchuan (8, 25) Ismail, Sukayna (7, 18)
Jacobberger, James W (9, 29) Jurkiw, Tom J (37)
Kalman-Maltese, Vivian (70, 71) Kane, Daniel P (6, 16)
74
Midwest DNA Repair Symposium May 17-18 Wayne State University
Kemp, Michael G (10, 33) Keri, Ruth (9, 29)
Khan, Shehnaz (71) Khasawneh, Rawia (65)
Kim, Christine (45) Kim, Dong Hyun (8, 25)
Kim, Sung-A (8, 25) Kim, Tae Moon (8, 25)
Kirkconnell, Killeen S (46, 53) Koncar, Robert (10, 31)
Kool, Daniel (36) Korolev, Sergey (61)
Kothandapani, Anbarasi (43) Kuan, Shih-Fan (7, 20)
Larson, Erik (56) Li, Guo-Min (45)
Liang, Zhuobin (10, 32) Liu, Guo (8, 21, 57, 69)
Liu, Shengqin (35, 40) Ljungman, Emily A (53)
Ljungman, Mats (10, 34, 46, 53) Logan, Hillary (8, 22)
Lord, Richard L (60) Magnuson, Brian (10, 34, 46, 53)
Mahmoud, Bassel (65) Mao, Guangzhao (54)
Marchal, Kate (71) Martin, Alberto (51)
Mason, Jennifer M (8, 22) McNally, Justin R (48)
Meetei, Amom Ruhikanta (10, 30) Mellon, Isabel (64)
Miller, Erin L (9, 27) Mirmoghtadaei, Nima (40)
Mishra, Akaash (44, 47) Montagna, Cristina (8, 25)
Morgan, Mary J (41) Mosel, Adam (35, 40)
Nallasivam, Sivakumar (10, 32) Nassar, Nicolas (10, 30)
Noronha, Anne M (36) Oakley, Greg G (35, 40)
O'Brien, Patrick J (9, 27, 37, 42, 48) O'Connor, Kathleen L (58)
O'Flaherty, Derek K (36) Oomen, Jerry (10, 34)
Orren, David K (64) Ortega, Janice (45)
Pan, Xinlei (9, 26) Park, Jung-Young (10, 30)
Park-York, MieJung (7, 19) Patrick, Steve M (43, 70, 71)
Paulsen, Michelle T (10, 34, 46, 53) Pence, Matthew G (9, 28)
Peng, Aimin (35) Pentz, Kyle (49)
Pisano, MM (59) Prasad, Jayendra (10, 34, 53)
Price, Carolyn (8, 23) Qian, Jiang (49, 50)
Rahassi, El Mustapha (10) Rajendran, Sountharia (68)
Rhoades, Nick A (66) Romick-Rosendale, Lindsey (10, 31)
Ryzhikov, Mikhail (61) Saadat, Nadia (54)
Samarajeewa, Dilini A (66) Sancar, Aziz (10, 33)
Sanders, Matthew (8, 24) Sarver, Jeffrey (70)
Sauls, Pegan (67) Sawant, Akshada (43)
Shalhout, Sophia (51) Sharma, Nidhi (49, 50)
Sharp, Kevin J (67) Shcherbakova, Polina V (6, 16)
Shekhar, Malathy (8, 9, 24, 54) Shelton, Brent (64)
Shiu, Patrick K T (66) Shriver. Storm J (36)
Silvestrov, Pavel (62) Singh, Amanpreet (38)
Singh, Thiyam R (10, 30) Smith, Christopher E (52)
Smith, SC (59) Smith, Zach (67)
Sobol, Robert (43) Son, Mi Young (8, 25)
Sosin, Angela (51) Srivastava, Amit Kumar (7, 17)
Stevens, Joshua B (8, 21, 57, 69) Stewart, Jason (8, 23)
75
Midwest DNA Repair Symposium May 17-18 Wayne State University
Sweasy, Joann (4, 6) Tateishi, Satoshi (8, 25)
Taylor, Mark R (37) Tong, Dan (45)
Tran, Amanda (9, 29) Turchi, John J (44, 47, 55, 71)
Unnikrishnan, Archana (7, 18, 65) VanderVere-Carozza, Pamela (55)
Veloso, Artur (10, 34, 46, 53) Ventrella-Lucente, Lisa (7, 18)
Wang, Ling (35) Wang, Qi-En (7, 17, 49, 50)
Wani, Altaf A (7, 17, 49, 50) Wani, Gulzar (49, 50)
Weatherford, Layne (63) Webb, C (59)
Weichselbaum, Ralph R (8, 22) Wells, Susanne (10, 31)
Whetstone, Ryan (7, 20) Wilds, Christopher J (36)
Williams, Jonathan (56) Wilson, Thomas E (10, 32, 34, 46, 53, 68)
Woods, Derek S (44, 47, 71) Worth, Leroy (11)
Wu, Megan (8, 22) Xiao, Hua (66)
Xu, Yong-jie (38) Ye, Christine J (8, 21)
Ye, Karen J (8, 21, 69) Yew, P. Renee (8, 25)
Yuan, Shuai (9, 28) Zhang, Fan (10, 30)
Zhang, Lei (9, 29) Zhang, Yanhua (54)
Zhang, Yaru (42) Zhang, Yowei (9)
Zhao, Linlin (9, 28, 36) Zhao, Ran (7, 17, 50)
Zhitkovich, Anatoly (43) Zhu, Qianzheng (49, 50)
Zielske, Steven (72)
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