Homologous Recombination & Double-Strand Break Repair: Crossing-Over with Cancer Biology

Homologous Recombination & Double-Strand Break Repair:Crossing-Over with Cancer Biology

Scott MorricalDept. of [email protected]

1. Lessons from Prokaryotes & Yeast2. DSBR in Humans-- Mediators, Paralogs, & BRCA1/2

mailto:[email protected]

Types of Recombination:

1. Site-specific recombination.Recombination occurs at defined, short sequences in DNA. Requires a site-specific recombinase enzyme that recognizes the target sequence.

2. Non-homologous or illegitimate recombination.Little or no sequence specificity or homology requirement.Certain types of transposition; non-homologous end joining.

3. General or homologous recombination.Occurs between any homologous DNA sequences of sufficientlength. Meiotic crossing-over; DNA repair.

Homologous Recombination & Cancer: Why You Should Care

1. Homologous recombination is required for accurate repairof DNA double-strand breaks (DSBs). Therefore protectiveagainst carcinogenesis. Errors increased mutation rates& susceptibility to carcinogenesis.

Nijmegen chromosome breakage syndrome (NBS) Ataxia telangiectasia (AT)AT-like disorder (ATLD) Bloom’s syndrome (BLM)Fanconi's anemia (FA) Werner’s syndrome (WRN)

2. Functions of human breast/ovarian cancer susceptibility genes BRCA1 & BRCA2 are clearly linked to homologous recombination and double-strand break repair.

3. Aberrant recombination phenotypes associated with neoplastic states-- i.e. hyper-recombination in p53 mutants.

4. Homologous recombination & DSBR are mechanisms oftumor cell resistance to radiation and chemotherapy. Targetingrecombination pathways in tumor cells could increase efficacy.

5. Targeted homologous recombination is desirable for cancergene therapy approaches, i.e. introduction of suicide genes atbenign locations in the genome.

Holliday Modelof HomologousGeneticRecombination

Mitotic Recombination:Double-Strand Break Repair Model

ZAP!!

Broken Chromosome

Undamaged Homologous Chromosome

Nucleolytic Processing

DNA Strand Exchange (HR)

DNA Synthesis (RDR)

Repaired Chromosome

Endonucleolytic Resolution & Ligation

3’

3’

3’

3’

Recombination Lessons fromProkaryotes:

The E. coli RecA Paradigm

XRCC3

hRAD51B

hRAD51D

hRAD51C

UvsXRecA

hRAD51

hDMC1

RadA

XRCC2

Pf

Ec

Os

Ll2

Dr

Yp2

Uu

RadB

Pf

T4

Structure, Function & Evolution of DNA Repair Enzymes

Phylogenetic Diversity of RecA Family

RB69

Types of DNA Rearrangements Catalyzed by E. coli RecA

2-strand reannealing:

+ATP ADP

3-strand exchanges:

ATP ADP+

ATP ADP

4-strand exchanges:

+ATP ADP

+

+ +

Properties of E. coli RecA Protein• Protomeric m.w. = 38 kDa.• Binds cooperatively to ssDNA at neutral pH; complex

stabilized by (d)ATP or ATPS, destabilized by ADP.• dsDNA binding requires low pH, ATPS, or transfer or

nucleation from ssDNA.• Forms filaments on & off of DNA.• Presynaptic filament-- RecA filament assembled on ssDNA in

presence of Mg(d)ATP-- is catalytically active form.• Catalyzes DNA-dependent (d)ATP hydrolysis.• Catalyzes (d)ATP-dependent DNA rearrangements including

complementary strand reannealing & homologous 3- or 4-strand strand exchanges.

• Co-protease: In response to DNA damage, facilitates auto-proteolytic cleavage of LexA repressor which induces theSOS response in E. coli.

Electron Micrograph of Relaxed Circular dsDNA Molecule Coatedwith RecA Protein in Presence of ATPS

• Open, right-handed helical filament• DNA is markedly extended and underwound

Story et al.: X-ray Crystallographic Structure of E. coli RecA-ADP Complex (Single Subunit Shown)

Presynaptic Filaments• RecA crystallizes as helical polymer even w/o DNA• DNA binding loops L1 & L2 are disordered

The RecA Paradigm of Homologous Strand Transfer

HomologousdsDNA

RecA

ATP, SSB

+

ssDNA

5’

3’

ATPADPATPADP

Other Recombination Proteins AffectDNA Strand Exchange

• Nucleases/helicases generate ssDNA substrates for presynapsis.

• ssDNA-binding proteins (SSBs)-- promote presynapsis*,sequester displaced strand in branch migration.

• Recombination mediator proteins (RMPs)-- assemble presynaptic filament.

• DNA helicases/translocases-- promote branch migration,filament remodeling.

Problems in Presynaptic Filament Assembly:

• Targeting filament assembly onto ssDNA in the presence of excess cellular dsDNA.

• Competition between RecAs and abundant cellular SSBs for binding to ssDNA.

Order of Addition Effect:-- SSB added to ssDNA after preincubation of RecA + ssDNA + ATPgives optimal stimulation of filament assembly, ATPase, & strandexchange activities.-- SSB preincubated with ssDNA before RecA + ATP added givesstrong inhibition of filament assembly, ATPase, & strand exchange.

Both problems dealt with by RecombinationMediator Proteins (RMPs) & other factors

T4 phage E. coli S. cerevisiae H. sapiens

Recombinase: UvsX RecA Rad51 Rad51

SSB: Gp32 SSB RP-A RP-A

Mediator(s): UvsY RecO/R Rad52 Rad52RecF? Rad55/57 Rad51B,C,D?

Xrcc2,3?Brca2?

Evolutionary Conservation of Recombinase, SSB,& Mediator Functionalities

T4 UvsX-ssDNAPresynapticFilaments

Gp32

UvsX

Enzymology of DSBR

• Yeast RAD52 Epistasis Group

• Human Rad51 paralogs

• The BRCA connection

How (Unprogrammed) DNA Double-Strand Breaks Occur

1. Ionizing Radiation (i.e. X- & -rays) and some chemicalagents locally disrupt the backbones of both strands of B-form DNA.

2. Inappropriate cleavage of dsDNA by an endonuclease.

3. BER or NER enzyme processing of interstrand crosslinks orof base lesions too close to nicks on the opposite strand.

4. Replication fork collapse:--Replication past a single-strand disruption or nick.--Replication fork collisions with cleavage complexes of type I & II topoisomerases.

5. Deoxyribonucleotide starvation.

Implications for Cancer Treatment

• Radiation: Hope that rapidly proliferating tumor cells won’t beable to repair induced DSBs fast enough, & therefore selectivelyundergo apoptosis. Problems-- resistant cells are good at DSBR; doesn’t work well for slower-growing tumors; secondary effects.

• Topoisomerase poisons: Stabilize topo-DNA cleavage complexes,increase frequency of replication fork collapse in rapidly proliferatingtumor cells.

+ +nick

Topo-I+ camptothecin

+

Topo-II+ m-AMSA

?

• Hydroxyurea: Inhibitor of ribonucleotide reductase. Chemotherapy deprives rapidly proliferating tumor cells ofdeoxyribonucleotide precursors for DNA synthesis & repair.

Observation: DSBs accumulate in treated cells- why?

Many stalled replication forks; get converted intomitotic DSBs (?)

Inability to complete replicative steps of DSBRpathways.

RAD52 Epistasis GroupIn Yeast (& Humans)

Genetically Implicated in Homologous Recombination& Double-Strand Break Repair

Mitotic Recombination:Double-Strand Break Repair Model

ZAP!!

Broken Chromosome

Undamaged Homologous Chromosome

Nucleolytic Processing

DNA Strand Exchange (HR)

DNA Synthesis (RDR)

Repaired Chromosome

Endonucleolytic Resolution & Ligation

3’

3’

3’

3’

RAD52 Epistasis Group: Genes & Gene Products(All conserved in humans in one way or another)

Processing:MRE11 Mre11/Rad50/Xrs2 complex (MRX)RAD50 implicated in nucleolytic resection ofXRS2 (NBS1) DSBs 3’ ssDNA tails

Recombination:RAD51 Ortholog of E. coli RecARAD52 Mediator, annealing & strand exchange proteinRAD54 Snf2/Swi2 ATPaseRAD55 Rad51 paralogs; Rad55/Rad57 dimer = mediatorRAD57 (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3)RAD59 Rad52 paralogRDH54 Rad54 paralogRFA1 Lg. Subunit of RPA (SSB) heterotrimer

Rad52

Mediator

Single-strandAnnealing

Rings &Oligomers

“7-11”

StrandExchange

Yeast Rad52 relieves RPA order ofaddition effect in Rad51-catalyzed DNA strand exchange assay…

RPA -> Rad51

Rad51 -> RPA

RPA+Rad52 -> Rad51

…but Rad52 does not replace RPAin strand exchange; rxns remainRPA-dependent.

72 min

36 min

Mediator FunctionOf Yeast Rad52

Biochemical Demonstration of Yeast Rad51-Rad52 Interactions

Immunoprecipitations:From wt extracts Affinity Chromatography:

From Rad52 overexpresser

Sung et al.

N-terminal Fragment of Human Rad52 (Residues 1-209) PromotesReannealing & Crystallizes as an Undecameric Ring

Singleton et al.; Kagawa et al.

I II

Propagation of Putative ssDNA Binding SiteAround the Ring Surface of HsRad521-209

Yeast Rad54• Member of Snf2/Swi2 family of DNA-dependentATPases/motor proteins/helicases.

• Binds to dsDNA and introduces local and global changes in superhelicity consistent with translocation along duplexwithout unwinding.

• Binds to Rad51 and stimulates DNA strand exchange rxns.

• Overcomes dsDNA inhibition of Rad51-catalyzed DNAstrand exchange.

Rad51 differs from E. coli RecA in having an intrinsically high affinity for dsDNA-- the dsDNAcan actually sequester Rad51 & thereby inhibitstrand exchange initiated from ssDNA.

Heyer & co-workers:

Rad54 Disassembles Inappropriate Rad51-dsDNA Complexes& Thereby FacilitatesAppropriate ssDNA-Initiated DNA Strand Exchanges

Rad54 may alsopromote nucleosomerearrangementsaround target sequencein homologousduplex.

Mre11/Rad50/Xrs2 (MRX) Complex(Yeast & Human Versions)

• Localizes with nuclear “repair foci” following cellexposure to ionizing radiation.

• Implicated in resection of DSBs into 3’ ssDNA tails-- curious, since Mre11 is a weak 3’ 5’ exonuclease!-- Mre11 also has ssDNA endonuclease activity-- all Mre11 nuclease activities Mn++ dependent

• Mre11 & Rad50 are conserved in all kingdoms of life. Xrs2 is only weakly conserved. Human Mre11/Rad50 complex associates with Nbs1, which is deficient in NBS, a rare cancer-prone syndrome. Hypomorphic alleles of Mre11 cause A-TLD, a human chromosomal instability syndrome.

Proposed Role of MRX in DSB Resection in Yeast

Wild-type MRX: weak unwinding activity or recruits helicase.mre11-H125N: still unwinds but lacks ssDNA endonuclease.

Mre11

Rad50+

Mre11(2:2)

(2:1)

Anderson et al., J. Biol. Chem., Vol. 276, Issue 40, 37027-37033, October 5, 2001

ElectronMicroscopyOf YeastRad50-Mre11Complexes

Rad50: Member of SMC Family. Walker A & B ATP-Binding Motifs Separated by Long Coiled-Coil Domain, Used (?) to Orient Mre11 Subunits & Link DSB Sites

Human (Vertebrate)Rad51 Paralogs:

Rad51BRad51CRad51DXrcc2Xrcc3

Implicated in Homologous Recombination& Repair; Formation of Nuclear Rad51 Foci

Following IR Exposure, Etc.

Rad51B Knockouts in Chicken B Lymphocyte DT40 CellsCompromise Rad51 Repair Foci Induced by DNA Damaging Agents

Takata et al.

Human Rad51 Paralogs FormTwo Distinct Complexes:

BCDX2

CX3

(West, Sung, & other labs)

BCDX2-- 1:1:1:1 Stoichiometry

CX3-- 1:1 Stoichiometry

Summary:Biochemical Activities Ascribed to Rad51 Paralog Assemblies

& Sub-Assemblies

*Caution necessary since C and CX3 have weak strand exchange activities.

Complex

BCDX2BCDX2BCDX2X3CX3

ss-Binding

X (+ HJ, duplex)

XX

XX (+ duplex)

X (+ nicks)

X

ATPase

XXX

X

X

Mediator*

X (Rad51 ATP/ADP exchange) X

Strand Ex*

X X (no ATP?)

X (no ATP?)

X (no ATP?)

Role of Breast/Ovarian CancerSusceptibility GenesBRCA1 & BRCA2

In Homologous Recombinatin& DNA Repair

Nobody Said It Would Be Simple…

… But Evidence Suggests Brca2 Plays a Direct Role and Brca1 anIndirect Role in Promoting Rad51-Dependent Recombinational Repair

Brca1 Knockout Reduces Efficiency of Rad51 Repair FociFollowing Cisplatin or IR Exposure of Mouse ES Cells

Bishop & co-workers

IR-Induced Rad51 Foci Formation Requires Brca2(Spontaneous Rad51 Foci That Occur During S-Phase Are Brca2-Independent)

Cells contain Brca2mutant lacking nuclear localizationsignal; Brca2 stays in cytoplasm.

West

X-ray Structure of Mouse/RatBrca2 ssDNA-Binding DomainComplexed to Dss1 & ssDNA

Yang et al. (2002) Science 297, 1837-1848

Structure ofMouse Brca2DNA-BindingDomain:

D = Intact DBD-Dss1 complex

E = Tower deletion DBDmutant bound to Dss1 & oligo dT9

OB-fold: Oligonucleotide/oligosaccharide binding fold,structurally conserved.

Brca2DBDTower-Dss1-dT9 Complex at 3.5 Å5 of 9 ssDNA Residues Resolve, Bound Across OB2 & OB3

X-ray Structure of Human Rad51RecA Homology Domain

Complexed to Brca2 BRC Repeat

Pellegrini et al. (2002) Nature 420, 287-293

1.7 Å Structure of Human BRCA Repeat 4 (A.A. 1517-1551)Bound to RecA Homology Domain of Rad51 (S95 - C-Terminus)

An Ingenious Trick: BRC4 fused to N-terminus of truncated Rad51 via flexible linker-- suppresses natural tendency of Rad51 to self-aggregate!

BRC4

BRC4Rad51

Rad51

HsRad51vs.

EcRecA

Brca2 Inhibits Rad51 Filament FormationCrystallographic EcRecA Filament

Superposition of BRC4 (from Rad51-BRC4 structure) on a subunit of EcRecA filament shows BRCA4 at interface between 2 EcRecA subunits.

EcRecA interface Rad51-BRC4 interface

EcRecA sequence26-IMRL-29 mediatespolymerization by anti-parallel -strand pairing

Brca2 sequence 1524-FHTA-1527 interactswith Rad51 by anti-parallel -strand pairing

Brca2: Designed to Load Rad51 Onto ssDNA?

Multiple BRC RepeatsIn Brca2 Could Serveas a Pre-Loading &Assembly Site for Rad51,All Ready for Transfer Onto ssDNA Bound toOB-folds in the DNABinding Domain

3HB Motif in TowerDomain: Tether Complexto Duplex Portion ofTailed DSB???

Why Are Defects MainlyAssociated With Tumorsof Breast & Ovary???

Other Cancer-Predisposition Syndromes:

1. Ataxia telangiectasia (AT)

Symptoms: -- progressive neuronal degeneration, loss of cerebellar function-- immunodeficiency, sterility, clinical radiation sensitivity-- 60-180x increase in malignancies (70% lymphomas and T-cell leuk.)

Cellular -- chromosomal breakage, telomere instability, radiosensitivity

Phenotype -- radioresistant DNA synthesis, defective cell cycle checkpoints-- dysfunctional apoptosis, reduced p53 response-- residual unrepaired DSBs

Defective -- ATM (ataxia telangiectasia mutated)

Gene -- 3056 a.a. ATM protein is member of phosphoinositol 3-kinase family-- master regulator in a signaling network responsible for coordinatingDSB repair, checkpoint functions, & other signaling processes thatpromote cellular recovery and survival

DSB-induced phosphorylation rxnsmediated by the ATMkinase that lead to transcriptional changes, implementation of cell cycle checkpoints, and execution of DNA repair processes.


2. Nijmegen breakage syndrome (NBS)

Symptoms: -- resembles AT but lacks ataxia and telangiectasia-- immunodeficiency, radiation sensitivity-- predisposition to malignancies

Cellular -- similar to AT

Phenotype

Defective -- NBS1

Gene -- 754 a.a. NBS1 protein is a component of Mre11-Rad50-Nbs1 complex that is implicated in processing DSBS into 3’ ssDNA tails.-- Phosphorylation at Ser343 and other sites by ATM kinase is necessary for IR resistance.

QuickTime™ and aTIFF (Uncompressed) decompressor

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Domain Structure of Nbs1

N-terminus FHA (forkhead-associated) / BRCT (Brca1 c-terminal) domainmediates association of Nbs1 with histone -H2AX, which is subsequently phosphorylated by ATM kinase.



Function of NBS1 in rejoining of DSBs and cell cycle checkpoint control.

NBS1 is recruited to damaged sites by binding to MRE11 in an ATM- independent manner and subsequently, the MRN complex recruits ATM kinase to such sites and H2AX is phosphorylated by ATM and othermembers of the ATM-related protein kinases.

The phosphorylation of H2AX recruits/retains more MRN complex at damaged sites, and initiates HRrepair.


3. Fanconi’s anemia (FA)

Symptoms: -- predisposition to malignancies, espeically acute myeloid leukemia(15,000x increase), squamous cell carcinoma (avg. onset age = 24 yrs)-- progressive aplastic anemia caused by loss of bone marrow stem cells-- diverse developmental abnormalities

Cellular -- chromosomal sensitivity to crosslinking reagents

Phenotype

Defective -- FancA, FancC, FancE, FancF, FancG --> nuclear complex

Genes -- FancD2 --> ubiquitination target; phosphorylated by ATM kinase-- FancB = FancD1 = BRCA2!!!!!



BRCA1 is required for ubiquitination of FANCD2. The activated FANCD2 protein is then seen to colocalize with BRCA1 in nuclear foci, where it may interact with other repair proteins. BRCA1 is known to interact with BRCA2 (FANCD1) which in turn interacts with the RAD51 recombinase. RAD51 protein is likely to play a direct role in DNA repair thus completing the cycle. Not shown: FANCB, which may also be related to BRCA2.

Interactions between the FA proteins and their potential roles in DNA repair.

DNA Mismatch Repair (MMR)Defects in Herditary

Non-Polyposis Colon Cancer(HNPCC)

Single base mismatches-- misincorporation by DNA polymerase,missed by proofreading exonuclease.





Insertion-deletion loops (IDLs)-- caused by polymerase slippage onrepetitive template, gives rise to Microsatallite Instability (MSI).


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E. coliMethyl-DirectedMismatch RepairSystem

Eukaryotic Homologs of MutS and MutL

Mlh1-Pms1

Heterodimers of Eukaryotic MutS & MutL Homologs

Msh2 Msh3

Mlh1-Mlh2

Msh2 Msh3

Mlh1-Mlh3

Msh2 Msh3

Mlh1-Pms1

Msh2 Msh6

Rad1-Rad10

Msh2 Msh3 Msh4 Msh5

Mlh1-Mlh3

Non-homologoustail removal inrecombinationintermediates

Insertion/deletionloop (IDL)

removal

Repair ofbase-base mismatches

Promotion ofmeiotic crossovers

MutS

MutS

MutL

MutL

*Note: This is yeast nomenclature.Mlh1 paralogs have different namesin yeast and humans.

1 b2-4 b

Model for Eukaryotic Mismatch Repair

HNPCC: Colon cancer predisposition syndrome, ~5% of all colorectal cancers

Early onset (~40-50 yrs), tumors typically of proximal colon, also with increasedrisk for developing tumors of endometrium, ovary, stomach, & small intestine.

Turcott’s syndrome (colorectal tumors & glioblastoma) and Muir-Torre syndrome(colorectal and skin gland cancers) share genetic features with HNPCC.

Microsatellite instability (MSI) found in mono-, di-, tri-, and tetranucleotiderepeat sequences in tumors taken from HNPCC patients.

MSI linked to defects in any of several MMR genes.



Models to explain rejection of heteroduplex intermediates containing mispairs via MMR proteins. In this figure, base pair differences between the recipient and donor chromosomes are indicated by the solid circles. (1) The mismatch correction process itself could lead to resection of nicked strands and the creation of a single-stranded gap that destroys the recombination intermediate. (2) hDNA rejection results in the unwinding of the annealed strands by a helicase that takes its cue from interactions with bound Msh factors. (3) Binding of MMR factors blocks attempted hDNA formation (Sugawara et al., unpublished).

Anti-recombination Effects of MMR Machinery

Observation: MutS, MutL, and to a lesser extent UvrD guard against homeologousrecombination between divergent DNA sequences. Mutations at these loci increaseHomeologous recombination frequencies by 100x to 1000x or more. Similar observationswith MMR mutants in yeast.



Two steps in recombination in which the Msh2p-Msh3p complex may interact with recombination intermediates. (Left) Msh2p-Msh3p loads onto DSB sites at recessed ends (1) and/or plays an active role in scanning hDNA and interacts with loops formed during pairing of homeologous sequences (2), leading to their rejection from the heteroduplex. (Right) Msh2p-Msh3p binds at the junction of homologous and nonhomologous DNA allowing for cleavage of unpaired tails by Rad1p-Rad10p (3) (adapted from reference 17).

Anti-homeologous recombination activity of MMR machinery maybe important for:

1. Speciation & evolution. Provides a barrier to inter-species recombination &thereby reinforces divergent processes.2. Prevents recombinational deletions of sequence-related genes and thereby stabilizes divergent gene duplications.3. These and other factors may contribute to tumor evolution.

Further Reading:

1. Recombinational DNA repair and human disease.Thompson & Schild (2002) Mutation Research 509, 49-78.

2. Mammalian DNA mismatch repair.Buermeyer et al. (1999) Annu. Rev. Genet. 33, 533-564.

3. Role of DNA mismatch repair defects in the pathogenesis of human cancer.Peltomaki (2003) J. Clinical Oncology 21, 1174-1179.

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Homologous Recombination & Double-Strand Break Repair: Crossing-Over with Cancer Biology