Slide 1Slide 2Slide 3Recombinational RepairHomologous RecombinationThe Double Strand Break Model for Recombination InitiationSlide 7Branch Migration of a Holiday JunctionSlide 9Slide 10Slide 11The Enzymes Responsible for Homologous Recombination in E. coliSlide 13Slide 14Slide 15Slide 16Slide 17RecA is Absolutely Required for All Homologous RecombinationSlide 19Slide 20Slide 21Clicker QuestionToo much repair?Recombination in meiosisOther types of recombinationRepair of DSBsDouble Strand Break Repair by Nonhomologous End Joining (NHEJ)Slide 28Slide 29NHEJSimultaneous DSBs can Result in TranslocationsMCB 250 Exam IIWednesday October 15th 7:00-9:00 PMSection Teaching Assistant RoomADQ, ADR, ADS, ADT, ADU, ADX, ADY, ADZSunetra Biswas, Preeti Ragunathan141 Loomis LaboratoryADE, ADF, ADG, ADH William Arnold 151 Loomis LaboratoryADA, ADB, ADC, ADV, ADM, ADOXinyun Cao, Michael Tencati114 David Kinely HallADI, ADJ, ADK, ADL, ADNTulip Mahaseth, Koh-Eun Narm100 Materials Science and Engineering (MSEB)MCB 250 Lecture 20Homologous RecombinationNonhomologous End JoiningRecombinational RepairReplication Through a SingleStrand Break Collapses the Replication ForkHomologous RecombinationRe-establishes Replication“Double strand breaks” (DSBs) can also be caused by:•Oxidative damage•Radiation damage•Cellular nucleasesHomologous Recombination•A DNA rearrangement - DNA is cut and rejoined. This can generate new combinations of genes: “recombination”.•“Homologous” means nearly identical.•Homologous recombination–is important for DNA repair (ds breaks (as we’ve seen) and other things).–is required for meiosis in eukaryotes: crossovers between homologous chromosomes required for proper chromosome segregation and for generating diversity in the gametes.–allows genes to be transmitted from one bacterial strain to a closely related strain. •Recombination takes place in all organisms - viruses, bacteria, archaea, eukaryotes.The Double Strand Break Model for Recombination InitiationFig 11-4DoubleStrand BreakProcessing toGenerate GappedDNA with 3’ TailsBabAbABaDouble StrandBreak ModelNote that RecombinationRequires DNA SynthesisFig 11-43’ Strand Invasion and PolI Mediated DNA SynthesisBranch Migration and Generation of Two Holiday JunctionsAaAaBabBbABbBranch Migration of a Holiday JunctionNote that the 3’ end invades the homologous duplexLike Fig 11-2StrandInvasionBranchMigrationHybridDuplexesBottomDuplexHolliday JunctionAacAaCcAaBbCcResolutionof a Holliday JunctionAaAacCAaCcBbCBbFig 11-3Resolutionof a Holliday JunctionCrossover PatchcCAacCAacCAacCCAaAaDNACleavagecFig 11-3A collapsed replication fork can be repaired by homologous recombination by a process very similar to that we’ve discussed in the previous few slides for DSB repair.The Enzymes Responsible for Homologous Recombination in E. coli•The recombination process is catalyzed by proteins. Several different proteins are required.•There is more than one pathway. Even in a particular pathway there may be more than one way to carry out individual steps. Some of the proteins required for recombination do other things also.•We will focus on what seems to be the primary pathway–RecA - required for all pathways–RecBCD–RuvABC–(Single Strand Binding Protein, PolI and Ligase)RecBCD generates a 3’ overhang to allow strand invasion.RecBCD –helicase/nuclease binds to ds break, helicase unwinds and nuclease degrades both strands.Fig 11-5When RecBCD encounters a chi site (5’GCTGGTGG3’) it’s activity changes. It stops degrading the strand with 3’ end and becomes more active degrading the strand with the 5’ end. This generates the 3’ tail needed to initiate recombination.Chi sites are dramatically overrepresented in the E. coli genome:Predicted: 1 / 65 kb Found: 1 / 4.5 kbFig 11-5RecA forms filaments on ssDNA.RecA Mediates Strand InvasionFor lots more examples of RecA catalyzed strand invasion reactions look at the rest of Fig. 11-8.Rec A + ATPRecA is Absolutely Requiredfor All Homologous Recombination•recA mutants do not do homologous recombination.•recA is conserved throughout evolution – the eukaryotic version is Rad51.•RecA initially binds to a single strand and forms a filament 5’ to 3’.•It then searches for homology and then initiates strand invasion and duplex formation.•RecA requires at least 50 bp of homology but works better with longer regions of homology.Resolutionof a Holliday JunctionAaAacCAaCcBbCBbRuvAB are Responsible for Branch Migration of Holliday JunctionsFig 11-17RuvB is an ATP-dependenthelicaseRuvA recognizes and binds to the Holliday junction.RuvC Cuts the Holliday JunctionRandomly Choosing Orientation – the Choice Determines if there is a “Crossover”Fig 11-18Clicker Question•Cytosine can spontaneously deaminate to Uracil•dCTP, a substrate for DNA synthesis, can also spontaneously deaminate to dUTP •Dut (dUTPase) gets rid of dUTP in the cell•Dut- RecA- double mutants are inviable•Which of the following additional mutations would you expect would suppress the Dut RecA phenotype?A.RecBCDB. MutSC. UngToo much repair?Increased U incorporation leads to increased attempted repairIn the absence of recombination this double stranded break is lethalUUUUUng and Base Excision RepairUUUUXDeathRecombination in meiosisHomologous chromosomes must pair and undergo recombination prior to 1st meiotic division. 1) Pairing and recombination ensure proper alignment and segregation to daughter cells and, 2) generate diversity (arrangements of genes different from either parent) in the final products of meiosis.This is initiated by creating double stranded breaks!Other types of recombination•Site-specific recombination –Specific short sequences in both donor and recipient and special enzymes can direct recombination between sequences with little or no homology. Can occur at high frequency.•Transposition–Requires the ends of the transposable element and special enzymes. Allows the transposable element to be inserted at many different sites without requiring homology.•Illegitimate recombination–Very low frequency recombination between non-homologous sequences.•Site-specific recombination and transposition will be discussed in the second half of this course.Repair of DSBs•Homologous recombination (HR) uses the information in an intact double stranded DNA molecule to repair the broken DNA molecule. HR can repair a DSB perfectly.•In