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Gross Chromosomal Rearrangements (GCRs)Visualized by SKY Analysis
Padilla-Nash, H.M. et al.; Genes, Chromosomes & Cancer. 25, 53 (1999)
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
GCR Assay
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
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
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
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
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
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.
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)
GenomeInstabilitySectionGMBBNHGRINIH
?
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
Genome-Wide ScreeningOf GCR Mutator Genes
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)
Canavanine plate
Canavanine & 5FOA plate
YPD plate
Group I : CAN & GCR mutator
Group II : CAN mutator
Group III : GCR mutator
Mutators Increased the Number of Resistant Colonies
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%)
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
The GCR Suppression by Ku70 and Ku86
Gross Chromosomal Rearrangements were observed in Human Ku86+/- Cell Line
Chromosome Fusion Ring Chromosome
GCRs observed in Human Ku86 +/- Cell Line
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