DNA Recombination IIBiochemistry 302Core interactions at junction crossover(A6C7C8 or R6-C7-Y8 motif)E. coli proteins participating in homologous recombinationBiochemical properties of RecA (Rad51)Biochemical and mechanistic features of RecA-mediated DNA recombinationAnother key player in homologous recombination:RecBCD enzymeProteins required for resolution of Holliday junctionsPutative roles of RuvA and RuvB proteins in branch migration (molecular spooling of DNA by twin “pumps”)In vitro assay for homologous pairing: hRad51 requires help (P. Sung lab)…but RPA (SSB) has an important role tooHomologous recombination and its utility for DNA repairRecA regulates the expression of genes involved in DNA repairBRCA2 enhances RAD51-dependent strand exchange in vitroEssential role of mediators in presynaptic filament assemblyFeatures of site-specific recombinationExample of site-specific recombination ? phage lysogeny (incorporation of ? genome into E. coli chromosome)Formation of intasome requires combined action of IHF and integraseOther types of gene rearrangements that use a recombination-like strategyDNA Recombination IIBiochemistry 302Bob KelmFebruary 4, 2004Core interactions at junction crossover(A6C7C8or R6-C7-Y8motif)Na+cavity formed by two A6 phosphates and bases of A6 and C7 nucleotidesF.A. Hays et al. (2003) JBC 278:49663B.F. Eichman et al. (2000) PNAS 97:3971parallel stacked-Xantiparallel stacked-Xopen-X (+ protein)WC H-bonds red dotsACC H-bonds blue dotsE. coli proteins participating in homologous recombination• Initiation– RecBCD nuclease (or exonuclease V)– other nucleases and RecQ helicase• Homologous pairing/strand exchange– RecA– other factors: SSB, RecF, and RecO, • Branch migration/junction resolution– RuvABCBiochemical properties of RecA (Rad51)• Structure– 38 kDa protein– Binds ssDNA as a homopolymer to form helical filaments (6 RecA monomers/turn)– DNA-dependent ATPase (strand exchange)• Function– Promotes pairing of homologous strands– Promotes 3 strand exchange process– Requires a free ssDNA end and DNA-DNA homology for strand invasionRecA (E.coli) ~ UvsX (T4) ~ Rad51Biochemical and mechanistic features of RecA-mediated DNA recombinationRecA-ssDNA filamentJoint moleculeneed ATPStrandexchange need ATP•RecA encircles ssDNA as a polymeric right-handed helical filament starting on 3′end.•RecA-ssDNA complex (w/ATP) searches dsDNA in a 5→3′ for complementary sequences capable of base pairing.•DNA is underwound and stretches to 1.5x its normal length to form joint molecule.•Once 3-strand intermediate is established, strand exchange and branch migration occurs (driven by ATP hydrolysis). •Strand exchange is catalyzed by RecA in vitro, but additional proteins are needed in vivo.Fig. 25-24Another key player in homologous recombination:RecBCD enzyme• Structure– Heterotrimeric protein complex – Proteins encoded by E. coli recB, recC, recD genes• Function (initiator of DNA recombination)– Multifunctional ATP-dependent endo/exonuclease and helicase having sequence specificity for Chi sites(crossover hotspot instigator, 5′-GCTGGTGG-3′, a sequence occurring frequently in the E. coli genome) – 1st:Helicase activity creates ssDNA loops after initial binding to double-strand break.– 2nd:Chi-stimulated nuclease activity generates a free 3′end to allow RecA-mediated strand invasion.Model depicting the functional roles of RecBCD, Chi sites, SSB, and RecA in initiating homologous recombinationFig. 25-28RecBCD jumping5′RecA recruitmentstrand switching at Chi sequenceRecA-mediatedRecA-SSB exchange5′3′3′5′Sister chromatidProteins required for resolution of Holliday junctions• RuvA– Involved in branch migration– DNA-binding protein with specificity for the four-stranded Holliday junction (tetramer)• RuvB– ATP-dependent motor protein/pump (hexamer)– Involved in branch migration via DNA spooling• RuvC– endonuclease– Begins resolution of Holliday junction (i.e. nicking/breaking of two strands)Putative roles of RuvA and RuvB proteins in branch migration (molecular spooling of DNA by twin “pumps”)Fig. 25-29In vitro assay for homologous pairing: hRad51 requires help (P. Sung lab)hRad51 mutant lacking ATPase activity required for rapid joint dissociationhRad54 augments hRad51-dependent strand exchangeS. Sigurdsson et al. (2002) JBC 277:42790…but RPA (SSB) has an important role tooReversal of ssDNA-mediated inhibition of DNA pairing and strand exchange by RPAS. Van Komen et al. (2002) JBC 277:43578Homologous recombination and its utility for DNA repair• Daughter strand gap repair – RecA-mediated– Other strand provides information to fill gap.• Double-strand break repair (eukaryotes)– Rad51, BRCA1?, BRCA2– RuvA, RuvB-like proteins• SOS or error-prone repair (mutation over death)– RecA, SSB, umuC, umuD allow Pol III (or Pol II) to copy past site of damage.Fig. 25-15What’s wrong with this picture?RecA regulates the expression of genes involved in DNA repair•RecA-ssDNA complex enhances proteolysis of LexA repressor and thereby activates the SOS regulon (unlinked genes regulated by a common mechanism).•dinA (DNA Pol II) dinB,F(damage-inducible genes)•urvA and uvrB (excision repair)•umuC,D (SOS error prone repair/mutagenesis)•sulA (cell division control)Fig. 26-32BRCA2 enhances RAD51-dependent strand exchange in vitroJoint moleculeNicked circleφX174 ssDNAφX174 dsDNAFig. 25-30Yang, H. et al. (2002) Science 297:1837Essential role of mediators in presynaptic filament assemblyP. Sung et al. (2003) JBC 278:42729BRCA2Paranemic (transient) joint formation….leads to transient opening of DNA strandsSearching for homologyP. Sung et al. (2003) JBC 278:42729Features of site-specific recombination• Protein-DNA dependent, no requirement for DNA homology – Exchange occurs between short (often complementary) recombination sites (~20-200 bp) or cis-acting sequences• Conservative– No DNA synthesis, no gain/loss of DNA• Requires a recombinase– Responsible for seeing cleavages sites or transient protein-DNA intermediates• Intermolecular or intramolecular– DNA inserted or excised (λ in E.coli)Example of site-specific recombination λ phage lysogeny (incorporation of λgenome into E. coli chromosome)Excision of prophage (Int, IHF, and Xis)Fig. 25-17Assisted by a bacterial protein, IHFDNA-bending factorsFormation of intasome requires combined action of IHF and