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Biochemistry of Recombinational DNA Repair

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Biochemistry of Recombinational DNA Repair BiochemistryBiochemistry ofof RecombinationalRecombinational DNADNA Repair:Repair: CommonCommon ThemesThemes StephenStephen KowalczykowskiKowalczykowski UniversityUniversity ofof California,California, DavisDavis •Overview of genetic recombination and its function. •Biochemical mechanism of recombination in Eukaryotes. •Universal features: steps common to all organisms. HomologousHomologous RecombinationRecombination AB ab+ Ab aB+ Genesis:Genesis: ScienceScience andand thethe BeginningBeginning ofof TimeTime How does recombination occur? And why? DNADNA ReplicationReplication CanCan ProduceProduce dsDNAdsDNA BreaksBreaks andand ssDNAssDNA GapsGaps RepairRepair ofof DNADNA BreaksBreaks Non-Homologous Homologous End-Joining (NHEJ) Recombination (HR) (error-prone) (error-free) dsDNA Break-Repair Synthesis-Dependent ssDNA Annealing (DSBR) Strand-Annealing (SSA) (SDSA) DoubleDouble--StrandStrand DNADNA BreakBreak RepairRepair 5' 3' 3' 5' + 3' 5' 5' 3' Initiation 1 5' 3' Helicase and/or 3' 5' nuclease 2 5' 3' 3' 3' 3' 5' Homologous Pairing & DNA Strand 3 Exchange 3' 5' 5' 3' RecA-like protein 5' 3' Accessory proteins 3' 3' 5' 4 3' 5' 5' 3' 5' 3' 3' 5' DNA Heteroduplex 5 Branch migration Extension proteins 3' 5' 5' 3' 5' 3' 3' 5' Resolution 6 Resolvase Spliced Patched ProteinsProteins InvolvedInvolved inin RecombinationalRecombinational DNADNA RepairRepair E. coli Archaea S. cerevisiae Human Initiation RecBCD -- -- -- SbcCD Mre11/Rad50 Mre11/Rad50/Xrs2 Mre11/Rad50/Nbs1 RecQ Sgs1(?) Sgs1(?) RecQ1/4/5 LM/WRN(?) RecJ -- ExoI ExoI UvrD -- Srs2 -- Homologous Pairing RecA RadA Rad51 Rad51 & DNA Strand SSB SSB/RPA RPA RPA Exchange RecF(R) RadB/B2/B3(?) Rad55/57 Rad51B/C/D/Xrcc2/3 RecO -- Rad52 Rad52 -- -- Rad59 -- -- Rad54 Rad54/Rdh54 Rad54/54B Brca2 DNA Heteroduplex RuvAB Rad54 Rad54 Rad54 Extension RecG -- -- RecQ Sgs1(?) RecQL/4/5 LM/WRN(?) Resolution RuvC Hjc/Hje -- -- -- -- Mus81/Mms4 Mus81/Mms4 ProteinsProteins InvolvedInvolved inin RecombinationalRecombinational DNADNA RepairRepair 5' 3' E. coli S. cerevisiae Human 3' 5' + 3' 5' RecBCD -- -- 5' 3' SbcCD Mre11/Rad50/Xrs2 Mre11/Rad50/Nbs1 Initiation 1 RecQ Sgs1(?) RecQ1/4/5/BLM/WRN(?) 5' 3' Helicase and/or 3' 5' nuclease RecJ ExoI ExoI 2 UvrD Srs2 -- 5' 3' 3' RecA Rad51 Rad51 3' 3' 5' Homologous Pairing SSB RPA RPA & DNA Strand 3 Exchange RecF(R) Rad55/57 Rad51B/C/D/Xrcc2/3 3' 5' RecF(R) Rad55/57 Rad51B/C/D/Xrcc2/3 5' 3' RecA-like protein 5' 3' Accessory proteins RecO Rad52 Rad52 3' 3' 5' 4 -- Rad59 -- 3' 5' -- Rad54/Rdh54 Rad54/54B 5' 3' 5' 3' -- -- Brca2 3' 5' DNA Heteroduplex 5 Branch migration Extension proteins RuvAB Rad54 Rad54 3' 5' 5' 3' RecG -- -- 5' 3' 3' 5' RecQ Sgs1(?) RecQL/4/5/BLM/WRN(?) Resolution 6 Resolvase Spliced Patched RuvC -- -- -- Mus81/Mms4(?) Mus81/Mms4(?) ProteinsProteins InvolvedInvolved inin RecombinationalRecombinational DNADNA RepairRepair 5' 3' E. coli S. cerevisiae Human 3' 5' + 3' 5' RecBCD -- -- 5' 3' SbcCD Mre11/Rad50/Xrs2 Mre11/Rad50/Nbs1 Initiation 1 RecQ Sgs1(?) RecQ1/4/5/BLM/WRN(?) 5' 3' Helicase and/or 3' 5' nuclease RecJ ExoI ExoI 2 UvrD Srs2 -- 5' 3' 3' RecA Rad51 Rad51 3' 3' 5' Homologous Pairing SSB RPA RPA & DNA Strand 3 Exchange RecF(R) Rad55/57 Rad51B/C/D/Xrcc2/3 3' 5' RecF(R) Rad55/57 Rad51B/C/D/Xrcc2/3 5' 3' RecA-like protein 5' 3' Accessory proteins RecO Rad52 Rad52 3' 3' 5' 4 -- Rad59 -- 3' 5' -- Rad54/Rdh54 Rad54/54B 5' 3' 5' 3' -- -- Brca2 3' 5' DNA Heteroduplex 5 Branch migration Extension proteins RuvAB Rad54 Rad54 3' 5' 5' 3' RecG -- -- 5' 3' 3' 5' RecQ Sgs1(?) RecQL/4/5/BLM/WRN(?) Resolution 6 Resolvase Spliced Patched RuvC -- -- -- Mus81/Mms4(?) Mus81/Mms4(?) Rad52,Rad52, Rad54,Rad54, Rad55,Rad55, andand Rad57Rad57 ProteinsProteins InteractInteract withwith Rad51Rad51 ProteinProtein andand StimulateStimulate DNADNA StrandStrand ExchangeExchange Rad54 Rad52 Rad55 Rad51 Rad57 RPA hRad51B/C/D hXRCC 2/3 hBRCA2 TheThe Rad51Rad51 NucleoproteinNucleoprotein FilamentFilament ATP ADP Yang, S., Yu, X., Seitz, E.M., Kowalczykowski, S.C., and Egelman, E.H. (2001) J. Mol. Biol., 314, 1077-1085 WhyWhy AreAre SoSo ManyMany ProteinsProteins RequiredRequired toto MakeMake aa RecA/Rad51NucleoproteinRecA/Rad51Nucleoprotein Filament?Filament? Answer: ssDNA Binding Protein (SSB/RPA) TheThe TemporalTemporal OrderOrder ofof PresynapticPresynaptic ComplexComplex FormationFormation 3’ Processed dsDNA with 3’-ssDNA overhang RPA RPA-ssDNA complex Rad52 Rad52 binds to the RPA-ssDNA complex Rad51 [ Rad51 is recruited by Rad52 Rad51 uniformly coats ssDNA to form the presynaptic complex New, J.H., Sugiyama, T., Zaitseva, E., and Kowalczykowski, S.C. (1998) Nature, 391, 407-410 Kowalczykowski, S.C. (2000). Some assembly required…. Nature Struct. Biol., 7, 1087-1089 Rad52Rad52 ProteinProtein HasHas TwoTwo FunctionsFunctions 1) Rad52 is a “recombination mediator” protein: it promotes displacement of RPA by Rad51 to facilitate Rad51 nucleoprotein filament formation. 2) Rad52 anneals ssDNA that is complexed with RPA: it promotes ssDNA annealing (SSA) and “second-end” capture in DSBR. Rad52Rad52 MediatesMediates AnnealingAnnealing ofof RPARPA-- ssDNAssDNA ComplexesComplexes 100 DAPI 80 RPA Rad52 60 RPA and Rad52 40 Annealing, % 20 Heat-denatured No Rad52 plasmid DNA 0 0 5 10 15 20 Time, min SingleSingle--strandstrand DNADNA AnnealingAnnealing 3’ 3’ Rad52, RPA, Rad59, Rad1/Rad10 Rad52Rad52 ProteinProtein FacilitatesFacilitates bothboth DNADNA StrandStrand InvasionInvasion andand ssDNAssDNA AnnealingAnnealing RPA 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein CatalyzesCatalyzes AnnealingAnnealing ofof ssDNAssDNA RPA 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein CatalyzesCatalyzes AnnealingAnnealing ofof ssDNAssDNA RPA 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein FacilitatesFacilitates bothboth DNADNA StrandStrand InvasionInvasion andand ssDNAssDNA AnnealingAnnealing RPA 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein MediatesMediates Rad51Rad51 NucleoproteinNucleoprotein FilamentFilament FormationFormation RPA 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein MediatesMediates Rad51Rad51 NucleoproteinNucleoprotein FilamentFilament FormationFormation RPA 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein CanCan PromotePromote SecondSecond-- EndEnd CaptureCapture byby ssDNAssDNA AnnealingAnnealing 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein CanCan PromotePromote SynthesisSynthesis-- DependentDependent StrandStrand AnnealingAnnealing 3’ 3’ Rad52 Rad51 SSA DSBR Rad52Rad52 ProteinProtein CanCan PromotePromote SynthesisSynthesis-- DependentDependent StrandStrand AnnealingAnnealing 3’ 3’ Rad52 Rad51 SSA DSBR ProteinsProteins InvolvedInvolved inin RecombinationalRecombinational DNADNA RepairRepair 5' 3' E. coli S. cerevisiae Human 3' 5' + 3' 5' RecBCD -- -- 5' 3' SbcCD Mre11/Rad50/Xrs2 Mre11/Rad50/Nbs1 Initiation 1 RecQ Sgs1(?) RecQ1/4/5/BLM/WRN(?) 5' 3' Helicase and/or 3' 5' nuclease RecJ ExoI ExoI 2 UvrD Srs2 -- 5' 3' 3' RecA Rad51 Rad51 3' 3' 5' Homologous Pairing SSB RPA RPA & DNA Strand 3 Exchange RecF(R) Rad55/57 Rad51B/C/D/Xrcc2/3 3' 5' RecF(R) Rad55/57 Rad51B/C/D/Xrcc2/3 5' 3' RecA-like protein 5' 3' Accessory proteins RecO Rad52 Rad52 3' 3' 5' 4 -- Rad59 -- 3' 5' -- Rad54/Rdh54 Rad54/54B 5' 3' 5' 3' -- -- Brca2 3' 5' DNA Heteroduplex 5 Branch migration Extension proteins RuvAB Rad54 Rad54 3' 5' 5' 3' RecG -- -- 5' 3' 3' 5' RecQ Sgs1(?) RecQL/4/5/BLM/WRN(?) Resolution 6 Resolvase Spliced Patched RuvC -- -- -- Mus81/Mms4(?) Mus81/Mms4(?) TheThe Srs2Srs2 HelicaseHelicase PreventsPrevents RecombinationRecombination byby DisruptingDisrupting Rad51Rad51 NucleoproteinNucleoprotein FilamentsFilaments Presynaptic filament formation is also under negative control. Veaute, X., Jeusset, J., Soustelle, C., Kowalczykowski, S.C., Le Cam, E., and Fabre, F. (2003) Nature, 423, 309-312 Srs2Srs2 HelicaseHelicase DisruptsDisrupts Rad51Rad51 NucleoproteinNucleoprotein FilamentsFilaments formedformed onon ssDNA,ssDNA, butbut notnot onon dsDNAdsDNA ssDNA: dsDNA: Rad54Rad54 ProteinProtein BothBoth StabilizesStabilizes thethe Rad51Rad51--ssDNAssDNA NucleoproteinNucleoprotein FilamentFilament andand PossessesPossesses Rad51Rad51--StimulatedStimulated ChromatinChromatin--RemodelingRemodeling ActivityActivity Mazin, A.V., Alexeev, A., and Kowalczykowski, S.C. (2003). J. Biol. Chem., 278, 14029–14036. Alexeev, A., Mazin, A., and Kowalczykowski, S.C. (2003) Nature Struct. Biol. 10, 182-186 Rad54Rad54 ProteinProtein IsIs aa MemberMember ofof Swi2/Snf2Swi2/Snf2 FamilyFamily ofof ProteinsProteins Potential leucine zipper Potential NLS I IA II III IV V VI 898 aa Rad51 protein binding Helicase motifs ••dsDNAdsDNA--dependentdependent ATPaseATPase activityactivity ••NoNo DNADNA helicasehelicase activityactivity ••TopologicallyTopologically unwindsunwinds dsDNAdsDNA ••InteractsInteracts withwith freefree Rad51Rad51 proteinprotein ••StimulatesStimulates DNADNA pairingpairing byby Rad51Rad51 proteinprotein ReconstitutionReconstitution ofof MononucleosomesMononucleosomes WithWith DefinedDefined PositionsPositions 148 N1 168 N1’ 33 178 N2 130 278 N2’ 108 253 N3 Rad54Rad54 ProteinProtein FacilitatesFacilitates NucleosomeNucleosome MobilityMobility ATPATP HydrolysisHydrolysis isis RequiredRequired forfor RemodelingRemodeling TheThe Rad51Rad51--ssDNAssDNA NucleoproteinNucleoprotein FilamentFilament EnhancesEnhances NucleosomeNucleosome RemodelingRemodeling byby Rad54Rad54 M ssDNA: + Rad51-ssDNA: + Rad54: +++++ + N3 N2 N1 DNA A Stoichiometric Complex of Rad54 Protein and Rad51-ssDNA Nucleoprotein Filament is Optimal for Nucleosome Remodeling StimulatoryStimulatory
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