Most UV lesions are repaired by Nucleotide Excision Repair (NER)Stalled replication forks may be bypassed by alternative (bypass) DNA polymerases(REV1, REV3, RAD30)

TT

Bypass polymerases have a cost They are “error prone” on normal sequences

Replication

-Replication through ssDNA creates DSB

-DSB can arise spontaneously or by artificial means

-Ionizing radiation

-Mechanical force i.e. mitosis

-Incomplete action of topos

-endonucleases

-How does the cell deal with these DSB and what are possible outcomes???

Recombination as a source of genetic instability

A hallmark of cancer cells is their genetic instability

Most of these types of instability may be explained by various mechanisms of homologous and nonhomologous recombination

In all probability the initiating lesion on DNA that leads to these changes in chromosomes can be attributed to a double-strand break (DSB)

Loss of heterozygosityReciprocal and nonreciprocal translocationsDeletions TruncationsChromosome loss

Abdel-Rahman et al. PNAS 98: 2538

Genome instabilityin tumorcells

TruncationsTranslocationsInversionsDuplicationsAmplifications

When breaks in DNA cannot be repaired, bad things happen

Here, DT40 cells lack thekey recombinationprotein, Rad51

Stalled replication forks may “break” (probably they are cut by an endonuclease)

Replication forks often stall at highly repetative sequences

size

shape

Restriction fragment

Replication fork “regression” Formation of a Holliday junction

Extension of leading strandBranch migration reverses Holliday junction and allows bypass of the UV-lesion

Holliday junction formation

Stabilize Replication Forks

Holliday Junctions

• All base pairs made• Cross Point (Branch can move)• Resolved with or without help of factors

Holliday Junction

Replication fork “regression” Formation of a Holliday junctionRuvA, RuvB

Extension of leading strandBranch migration reverses Holliday junction and allows bypass of the UV-lesion

Holliday junction formation

B

Shape

Helicase

RuvA

GO TO: http://www.sdsc.edu/journals/mbb/ruva.html

RuvB

RuvC Necessary for resolution

• RuvA/B sufficient to resolve HJ with an end nearby

• RuvC cleaves symmetrically on 2 homologous DNA segments

The EMBO Journal (1997) 16, 1464–1472, doi: 10.1093/emboj/16.6.1464

Branch Migration

Resolution of a Holliday Junction

Holliday Junction

Cleavage of a Holliday junction at a stalled replication fork produces an intact template and a broken-ended molecule

BIR

Binding of Rad51

Strand invasion

5’ to 3’ exonuclease resection

Homology search

Re-establishment of a replication fork

BIR

Branch migration allows gap to be filled in

Another Holliday junction

Ends flipped over for easy viewing

STRAND INVASION

Basic strand exchange

Replication Restart Can Lead to SCE

Recombination as a source of genetic instability

A hallmark of cancer cells is their genetic instability

Most of these types of instability may be explained by various mechanisms of homologous and nonhomologous recombination

In all probability the initiating lesion on DNA that leads to these changes in chromosomes can be attributed to a double-strand break (DSB)

Loss of heterozygosityReciprocal and nonreciprocal translocationsDeletions TruncationsChromosome loss

Abdel-Rahman et al. PNAS 98: 2538

Genome instabilityin tumorcells

TruncationsTranslocationsInversionsDuplicationsAmplifications

Eukaryotic recombination machinery complex

• Large number of proteins necessary– Rad51, Rad52, 5 Rad51

paralogs (load Rad51)

– Rad54 – strand invasion

– BRCA1 and BRCA2

• Rad51, BRCA1/2 KOs lethal

• Rad51 paralogs – less SCE

HOMOLOGOUS RECOMBINATION

Gene Conversion w/o Crossover

Break-Induced Replication

Gene Conversion w/ Crossover

DSB

NONHOMOLOGOUS RECOMBINATION

New telomere formation

NH End-Joining

Single-strand annealing

Nonreciprocal translocation

Gene amplification

Chromosomes and Chromatids break other times than during replication

Repaired BIR and Gene Conversion

BIR – Non replicating cell

In humans. Telomerase is shut off at birth. Telomeres get shorter and shorter every time DNA replicates.People who have a deletion of one of the two telomerase RNA genes suffer from

dyskeratosis congenita (haplo-insufficiency)

Tumors arise by: (a) re-activating telomerase(b) ALT (alternative lengthening of telomeres)

ALT

• Extension telomeres by HR demonstrated yeast

• Deleted telomerase enzyme– Shorten 10nt/generation– Die 30-50 generations– Few telomerase independent survivors

• Eliminated Rad52

Gene Conversion - Synthesis dependent strand annealing (SDSA)

inducible

Requires Rad51, Rad52, Rad54 etc.

Also requires PCNA and DNA ploymerases

Cell knows which donor to choose – requires recombination enhancer

Restriction size differences at a and alpha site

Can look for presence of HJs• Studied using 2D Gels hotspots

• Looking for differences novel spots not part of replicating arc of DNA

• Cut out and use denaturing gel– Each strand characterized

HOMOLOGOUS RECOMBINATION

Gene Conversion w/o Crossover

Break-Induced Replication

Gene Conversion w/ Crossover

DSB

NONHOMOLOGOUS RECOMBINATION

New telomere formation

NH End-Joining

Single-strand annealing

Nonreciprocal translocation

Gene amplification

Chromosomes and Chromatids break other times than during replication

Repaired BIR and Gene Conversion

Hin gene

promoter

H1 gene Rh2 gene OP H2 gene

Hin gene

promoter

H1 gene Rh2 gene OP H2 gene

PP

No transcription

Site-specific Recombination

Nicks on one pair of strands

Reciprocal strand exchange(Holloiday junction)

Nicks on second pair of strands

Reciprocal strand exchange

Two examples: Hin and FLP

Site-specific recombination

One example: phase variation in Salmonella

Flagella composed of H1 flagellin

Anti H1 antibodyeliminates 99.99%

Survivors have alternativeH2 flagellin