Gross Chromosomal Rearrangements (GCRs) Visualized by …GMBB NHGRI NIH? S Phase Checkpoint M Phase Checkpoint tgtgtgtgtg acacacac de novo Telomere Addition Translocation GCRs Recombination

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