gross chromosomal rearrangements (gcrs) visualized by …gmbb nhgri nih? s phase checkpoint m phase...
TRANSCRIPT
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Gross Chromosomal Rearrangements (GCRs)Visualized by SKY Analysis
Padilla-Nash, H.M. et al.; Genes, Chromosomes & Cancer. 25, 53 (1999)
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1. Gross Chromosomal Rearrangements Assay
2. Rad1 and Rad10 in the Formation of GCR
3. Genome-wide Screening of GCR Mutator Genes
4. Recombination and Post Replication Repairfunction for the GCR formation.
5. Ku70/86’s Role in the Suppression of GCR
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GCR Assay
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Cans, 5FOAs
Canr, 5FOAr
Wild type rate of independent mutation = 10-12 to 10-14 per generation.
Wild type rate of genome rearrangements = 3.5 x 10-10 per generation.
Assay for Measuring the Rate of Gross Chromosomal Rearrangements (GCRs)
CAN1URA3 CEN5tgtg tgtg
CAN1URA3 CEN5tgtg tgtg
CEN5tgtg
or
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tgtg tgtg
Any Chr.
tgtg tgtg
Chr. VCAN1
URA3
GCRs Observed in Mutator Mutants
tgtgtgtg tgtg
Chromosome fusion
tgtgtgtgtgtg
Terminal deletion withde novo telomere addition
tgtg ( )
Deletion
tgtgtgtgtgtg
Translocation
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AGTAAATAAGACAGAtgggtgtgggtgtgg
Chr.VAAACAGCAAAGGCCA:CAGAACCGTATTCATCATTGTCATTATATT:CCATTTTCGCGTCTCChr.X
Chr.VATATTGGTATGATTG:CCCTTGGTGGTACTAATACTTGGCCGATTG:AACTTTTCATTTGGTChr.I
Chr.VCCCAGGAGCCTGGGG:TCCAGGTATAATATCTCCAGGAGCCTGGGG:GCCTGGCATTATCTCChr.XIV
Terminal Deletion & Telomere Addition
Rearrangement Breakpoints Structures
Non-homologyTranslocation
Micro-homologyTranslocation
HomeologyTranslocation
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RB Deletion
RBTATAGCTTTTA..(>135kb)..CATGAATTTAAACATAAA
BCR-ABL Translocation
BCRGATTAGCCAGGCTAGGCAGT:GGGCACCTGTAATCACAACTGCCAAAGTTTGTCTACCCAGT:TTTAAATCCTGGCTTTCCCCTABL
BCRCTCATCGGGCAGGGTGTGGG:GAAACAGGGAGGTTGTTCAGAAGTAAATTAAGGGTTATGGG:TCTTCACTTTCGTAGCTTCTAABL
From: J. G. Zhang et al., 1995; S. Canning & T. Dryja, 1989
Breakpoint Sequences in Cancer
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Sensing of DNA Damage
Generation of DNA Damage
GCR Formation
Process of DNA Damageto Proper Substrates of GCRs
DNA Repair
S phase checkpoint
?
Post-replication repair ?(RAD5/RAD18)
Break induced replication (BIR)
Telom
erase
PIF1
LIG4 + ?
CDC50 ? : Cell cycle regulation for suppression of GCRs ?
CSM2 ?
MUS81/MMS4 ?
ESC1 ? : activation of gene expression for suppression of GCR ? Silencing defect ?
RAD5 ?
RAD18 ?
TSA1 ?ALO1 ?
ELG1 ?
ELG1 ?
MUS81/MMS4 ?
MUS81/M
MS4 ?
CAC1/ASF1Mit
otic c
heckpo
ints
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Rad1 and Rad10
Radiation-sensitive mutants of Saccharomyces cerevisiae
Single-stranded DNA specific 5’-endonuclease
Heterodimer complex composed with Rad1 (~126 kDa, basic) and Rad10(~24 kDa, acidic).
The Rad1 and Rad10 are required for damage-specific recognition andincision of DNA during nucleotide excision repair. Bardwell L et al.,Mol Cell Biol. 12, 3041 (1992)
Rad1 and Rad10 also function in recombination. Schiestl RH andPrakash S., Mol Cell Biol. 8, 3619 (1988) & 10, 2485 (1990)
Human homologs are XPF (Rad1) and ERCC1 (Rad10) and XPF mutationshave been documented in Xeroderma pigmentosum.
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rad1 and rad10 Mutations Reduced the GCR Rate in GCR Mutator Strains
Genotype Wild type rad1 rad10
Wild type 3.5 x 10-10 3.8 x 10-10 3.5 x 10-10(1) (1) (1)
rfa1-t33 4.7 x 10-7 7.4 x 10-9 5.1 x 10-9(replication) (1343) (21) (15)mre11 2.2 x 10-7 3.9 x 10-8 6.0 x 10-8(Multifunction) (629) (111) (171)mec1 6.4 x 10-8 5.4 x 10-9 2.0 x 10-8(Checkpoint) (183) (15) (56)rad52 4.4 x 10-8 2.2 x 10-9 2.8 x 10-9(Recombination) (126) (6) (8)
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GenomeInstabilitySectionGMBBNHGRINIH
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S PhaseCheckpoint
M PhaseCheckpoint
tgtgtgtgtgacacacac
de novo TelomereAddition
Translocation
GCRsRecombination
CORRECT REPAIR
Rad
1/10
Rad1/10
GCRsTelomerase
Rad
1/10
Telomerase
Pif1
Lif1(XRCC4)
Lif1(XRCC4)Lig4
Rad1 and Rad10 Function to Produce BetterStructure for Spontaneous GCR Formation
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Genome-Wide ScreeningOf GCR Mutator Genes
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a HIS- G418r URA-Sporulation
Mating
KANX
MFA1 promoter-HIS3 (off)
CAN1 URA3
Gene X
α HIS- URA+
a HIS+ G418r URA+CAN1URA3
KAN
MFA1 promoter-HIS3 (on)
a
a
X
A Scheme for Screening of All Non-EssentialYeast Open Reading Frames for GCRs
G418r
(GCR assay strain)
(K/O library for screening)
(K/O library 4,644 clones)
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Canavanine plate
Canavanine & 5FOA plate
YPD plate
Group I : CAN & GCR mutator
Group II : CAN mutator
Group III : GCR mutator
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Mutators Increased the Number of Resistant Colonies
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Effect of New GCR Mutator GeneDefects on the GCR Rates
WT pif1-m2
Relevant
Genotype
GCR rate
(CANr-5FOAr)
GCR rate
(CANr-5FOAr)
Wild type 3.5 x 10-10 (1) 6.3 x 10-8 (180)
alo1! 4.7 x 10-8 (134) 1.1 x 10-7 (314)
cdc50! 4.8 x 10-9 (14) 2.6 x 10-7 (743)
csm2! 2.7 x 10-9 (8) 1.6 x 10-7 (457)
elg1! 1.7 x 10-8 (49) 3.0 x 10-7 (857)
esc1! 2.3 x 10-9 (7) 1.1 x 10-7 (314)
mms4! 5.9 x 10-8 (169) 2.3 x 10-7 (657)
rad5! 6.3 x 10-8 (181) 2.2 x 10-7 (633)
rad18! 7.1 x 10-8 (202) 2.5 x 10-7 (714)
tsa1! 2.6 x 10-9 (7) 3.6 x 10-7 (1029)
ufo1! 2.6 x 10-8 (74) 1.4 x 10-7 (400)
de novo telomere addition (100%)de novo telomere addition (100%)
de novo telomere addition (70%)+ translocation (30%)
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GenomeInstabilitySectionGMBBNHGRINIH
Sensing of DNA Damage
Generation of DNA Damage
GCR Formation
Process of DNA Damageto Proper Substrates of GCRs
DNA Repair
S phase checkpoint
Mitoti
c chec
kpoint
s
Post-replication repair ?(RAD5/RAD18)
Break induced replication (BIR)
Telom
erase
PIF1
LIG4 + ?
?
CDC50 ? : Cell cycle regulation for suppression of GCRs ?
CSM2 ?
MUS81/MMS4 ?
ESC1 ? : activation of gene expression for suppression of GCR ? Silencing defect ?
RAD5 ?
RAD18 ?
TSA1 ?ALO1 ?
ELG1 ?ELG1 ?
MUS81/MMS4 ?
MUS81/M
MS4 ?
CAC1/ASF1
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The GCR Suppression by Ku70 and Ku86
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Gross Chromosomal Rearrangements were observed in Human Ku86+/- Cell Line
Chromosome Fusion Ring Chromosome
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GCRs observed in Human Ku86 +/- Cell Line
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ACKNOWLEDGEMENTS
NHGRI/NIH
Soma BanerjeeAmalia Dutra
Ji-Young Hwang Akira MotegiEvgenia Pak
Stephanie Smith
Amitabha GuptaAnju Majeed
U. Minnesota
Eric HendricksonGoutam GhoshFarjana Fattah
Gang Li