Mechanisms of Homologous Recombination
Pratik ShriwasJohn Elmore
Quyen LuongMCB 7200
December 3rd 2015
Origins1900s : Certain genes are genetically linked1911 : Crossover occurs between linked
genes1931 : Crossover occurs during meiosis and
mitosis1947 : Genetic recombination in bacteria1964 : Proposed model of Holliday junction1980 and beyond: Several models of
Homologous recombination in different species Lobo and Shaw (2008) ; Matos and West (2014)
Homologous Recombination (HR)It is the biological process of genetic
exchange between two similar or identical nucleic acids
Archaea, Eukarya and Bacteria as well as viruses
Enzymes involved have homologous domains and are evolutionarily conserved
Mitosis, Meiosis and Horizontal gene transfer
Pérez-Losada, et. al. (2015); Matos and West (2014)
Bacteria Horizontal Gene
transferDNA repair
Integration of donor DNA into recipient cell
TransformationTransductionConjugation
UV or other radiation and chemical mutagens
Double stranded breaks
Vox, M. (2009) ; Hanada and Yamaoka (2014)
Eukaryotes Before Mitosis
MeiosisOccurs during cell
division after DNA replication
During Interphase (S and G2)
Mitotic crossoverRepair Double stranded
breaks (DSBs)
Occurs during prophase I
Chromosomal crossover Genetic diversity with
newer combination of genes
Walsh, C.S. (2015)
VirusesDNA - DNA recombination (DNA viruses) RNA - RNA recombination (RNA viruses) Genetic diversity – Viral evolution
Pérez-Losada, et. al. (2015)
More or few chromosomesDown Syndrome
Improper segregation and nondisjunction
Inefficient DSB repair
Cancer and other related diseases
Failed HR
Improper HR
Strachan and Read (2011) ; Walsh, C.S. (2015)
Double Stranded Break – Initiation
BLM
Mimitou, et al, 2008
DSB Resectioning – A Closer look
Mre11
Rad50
NBS1
CtIP
CtIP
MRN
MRN
BLM
BLM
Spo11
Mimitou, et al, 2008;
Presynaptic Complex
- RAD51
- RAD52
- RPA
- BRCA2
- RAD54
D-Loop
TIME
‘Strand Invasion’
Mimitou, et al, 2008; Kowalczykowski, 2015; Fillipo et al, 2008; Renkawitz et al, 2014
DSB – Three Possible Fates
Mimitou, et al, 2008
Double Holiday Junction=
MUS81-
EME1
GEN
1
BLM/YEN1
GEN
1
Non-Crossover Crossover products
MUS81-
EME1
Resolution
Mimitou, et al, 2008; Fillipo, et al, 2014; Kowalczykowski, 2015
HR ApplicationsTransgenic knockout animalsChimeric proteinsAnti-cancer therapy
Genetically Modified Organisms
Gene targeting knockout mice: Deliver artificial genetic material into mouse
embryonic stem cellsReplace the targeted genes with homologous
recombinationBreeding steps knockout mice
Homologous Recombination (HR)
Non-HR
Chimeric ProteinsSynthetic protein of two
proteins with >70% similarity
Structure and function are preserved
Point mutagenesis alters function with increasing amino acid substitution
Study of protein structure and function Carbone et al., 2007
Cancer TherapyNon-homologous end joining
(NHEJ) and HR to repair double-stranded breaks
NHEJ applies to all normal cells as well
Cells use HR to repair DNA double-stranded breaks resulted from anti-cancer treatment (e.g. radiotherapy)
HR inhibitors prevents tumor cells from repairing DNA breaks. Chernikova et al. 2012
Synthetic Lethality Inhibiting compensatory pathways in HR-
deficient tumor cells can increase cell death (increase effectiveness of treatment).
Poly(ADP-ribose) polymerase or PARP
DNA damage (Single-stranded breaks or base damage)
HR
Poly(ADP-ribose) polymerase or PARP
DNA damage (Single-stranded breaks or base damage)
HR
BRCA1/2 mutationsPARP inhibitors
Double Stranded Breaks
Double Stranded Breaks
Key Points1. Biological processes in species with HR,
improper HR2. RAD51 binds ssDNA and causes ‘strand
invasion’ D-loop formed Double Holiday Junction formed DNA polymerase fills gaps in break dHJ resolved to form either crossover or non-crossover products
3. HR can be used in gene manipulation, protein functional studies, and therapy for certain tumors.
4. Synthetic lethality can be used effectiveness of anti-cancer drugs
References1. Lobo, I., & Shaw, K. (2008). Thomas Hunt Morgan, genetic recombination, and gene mapping. Nature
Education, 1(1), 205.2. Matos, J., & West, S. C. (2014). Holliday junction resolution: Regulation in space and time. DNA repair, 19, 176-
181.3. Hanada, K., & Yamaoka, Y. (2014). Genetic battle between Helicobacter pylori and humans. The mechanism
underlying homologous recombination in bacteria, which can infect human cells. Microbes and Infection, 16(10), 833-839.
4. Vos, M. (2009). Why do bacteria engage in homologous recombination?.Trends in microbiology, 17(6), 226-2325. Walsh, C. S. (2015). Two decades beyond BRCA1/2: Homologous recombination, hereditary cancer risk and a
target for ovarian cancer therapy.Gynecologic oncology, 137(2), 343-350.6. Pérez-Losada, M., Arenas, M., Galán, J. C., Palero, F., & González-Candelas, F. (2015). Recombination in viruses:
mechanisms, methods of study, and evolutionary consequences. Infection, Genetics and Evolution, 30, 296-307.7. Strachan, Tom; Read, Andrew (2011). Human molecular genetics (4th ed.). New York: Garland
Science. ISBN 9780815341499. 8. Chernikova, S.B., Game, J.C., Brown, J.N. Inhibiting Homologous Recombination for Cancer Therapy. Cancer
Biology and Therapy, 13:2, 61-69, 20129. Carbone, M.N., Arnold, F.H, Engineering by homologous recombination: exploring sequence and function within
a conserved fold. Current Opinion in Structural Biology, 17:454-459, 200710. Kowalczykowski, S.C., An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring
Harbor Perspectives in Biology, 7:a016410, 201511. http://www.bio.davidson.edu/courses/genomics/method/homolrecomb.html12. Mimitou, E.P., Symington, E.P. Nucleases and Helicases take Center Stage in Homologous Recombination, Trends
in Biochemical Sciences. Vol. 34, No. 5, 200913. Renkawitz, et al. Mechanisms and principles of homology search during recombination, Nature: Molecular and
Cell Biology, 15: 369, 20414. Fillipo, J.S., Sung, P. Klein, H, Mechanism of Eukaryotic Homologous Recombination, Annual Review of
Biochemistry, 77:229-57, 2008