Molecular biology - Chapter 22: Homologous recombination

Molecular BiologyFourth EditionChapter 22Homologous RecombinationLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.122.1 Homologous Recombination Pathways2RecBCD Pathway – Initial BindingRecBDC-sponsored homologous recombination in E. coli:DNA helicase activity unwinds the DNA toward a Chi-siteSequence 5’-GCTGGTGG-3’Chi sites found on average every 5000 bp in E. coli genomeRecBCD protein has ds- and ss-exonuclease activityss-endonuclease activityActivities permit RecBCD to produce a ss-tail now coated by RecA protein3The RecBCD Pathway Schematic RecBCD pathway is a well-studied homologous recombination pathway used by E. coli4RecBCD Pathway – D LoopInvasion of a duplex DNA by a RecA-coated single-stranded DNA from another duplex that has suffered a double-stranded breakInvading strand forms a D loop (displacement)Loop is defined by displaced DNA strandWhen tail finds homologous region, nick occurs in in D-looped DNANick allows RecA and ss-break create a new tail that can pair with gap in the other DNASubsequent degradation of the D-loop strand leads to the formation of a branched intermediate5Holliday JunctionsBranch migration in this intermediate yields a Holliday junction with 2 strands exchanging between homologous chromosomesBranch in the Holliday junction can migrate in either direction by breaking old base pairs and forming new ones in a process called branch migrationThis migration process does not occur at a useful rate spontaneouslyDNA unwinding requiredUnwinding requires helicase activity and energy from ATP6Resolving Holliday JunctionsHolliday junctions can be resolved by nicking 2 of its strandsYielding: 2 noncrossover recombinant DNAs with patches of heteroduplex2 crossover recombinant DNAs that have traded flanking DNA regions722.2 Experimental Support for the RecBCD Pathway - RecAThe recA gene has been cloned and overexpressed with abundant RecA protein available for studyIt is a 38-kD protein that can promote a variety of strand exchange reactionsThere are 3 stages of participation of RecA in strand exchangePresynapsis – RecA coats the ss-DNASynapsis – alignment of complementary sequences in ss- and ds-DNAsPostsynapsis – ss-DNA replaces the (+) strand in ds-DNA to form a new double helixJoint molecule is an intermediate in this process8PresynapsisIn the presynapsis step of recombination:RecA coats a ss-DNA participating in recombinationSSB accelerates the recombination processMelting secondary structurePreventing RecA from trapping any secondary structure that would inhibit strand exchange later in the recombination process9Synapsis:Synapsis is the proper alignment of complementary sequencesSynapsis occurs when:Single-stranded DNA finds a homologous region in a double-stranded DNAThis ss-DNA aligns with the ds-DNANo intertwining of the 2 DNAs occurs at this point10Postsynapsis:Strand ExchangeRecA and ATP collaborate to promote strand exchange between ss- and ds-DNAATP is necessary to clear RecA off the synapsing DNAs This makes way for formation of ds-DNA involving the single strand and one of the strands of the DNA duplex11RecBCDRecBCD has a DNA endonuclease activityNicks ds-DNA especially near Chi sitesATPase-driven DNA helicase activity that can unwind ds-DNA from their endsThe activities help RecBCD provide the ss-DNA ends that RecA needs to initiate strand exchange12RuvA and RuvBRuvA and RuvB form a DNA helicase that can drive branch migrationRuvA tetramer with square planar symmetry recognizes the center of a Holliday junction and binds to itLikely induces the Holliday junction itself:To adopt a square planar conformationTo promote binding of hexamer rings of RuvB to 2 diametrically opposed branches of the Holliday junctionRuvB uses its ATPase to drive the DNA unwinding and rewinding necessary for branch migration13A Synthetic Holliday JunctionMix oligonucleotides at annealing conditions for complementary base-pairing5’-end of oligo 2 base-pairs with the 3’-end of oligo 15’-end of oligo 1 base-pairs with the 3’-end of oligo 2Ends cross over in pairing14RuvCResolution of Holliday junctions is catalyzed by the RuvC resolvaseThis protein acts as a dimer to clip 2 DNA strands to yield either patch or splice recombinant productsClipping occurs preferentially at the consensus sequence 5’-(A/T)TT(G/C)-3’Branch migration is essential for efficient resolution of Holliday junctionsEssential to reach preferred cutting sitesRuvA, B, and C work together in a complex to locate and cut those sites15Resolution of a Holliday JunctionHolliday junction can be resolved in 2 ways:Cuts 1 and 2 yield 2 duplex DNAs with patches of heteroduplex Cuts 3 and 4 yield crossover recombinant molecules with the 2 parts joined by a staggered splice1622.3 Meiotic RecombinationMeiosis in most eukaryotes is accompanied by recombinationThis process shares many characteristics with homologous recombination in bacteriaThis section focuses on meiotic recombination in yeast17Mechanism OverviewStart with chromosomal lesion: ds-DNA breakNext exonuclease recognizes the breakDigests the 5’-end of the 2 strandsCreates 3’-single strand overhangsOne single-stranded end can invade other DNA duplex, forming a D loopDNA repair synthesis fills in the gaps in the top duplex expanding the D loopBranch migration can occur in both directions leading to 2 Holliday junctionsHolliday junctions can be resolved to yield either a noncrossover or a crossover recombinant18Model of Yeast Recombination19The Double-Stranded DNA BreakDNA cleavage uses 2 Spo11Active site Tyr as OHAttack 2 DNA strands at offset positionsTransesterification reaction breaks phosphodiester bonds within DNA strands Creates new bondsNicking DNA strandsNicking is asymmetricYields 2 sizes oligosRelease of Spo11-linked oligos 12-37 nt long20DSB End ResectionResection occurs on both strands using prior nicksRecombinases load asymmetrically onto the newly created single-stranded regionsOne protein tags coated free 3’-end for invasion into homologous duplexThis leads to initiating Holliday complex formation21Creation of Single-Stranded Ends at DSBsFormation of the DSB in meiotic recombination is followed by 5’3’ exonuclease digestion of the 5’-ends at the breakDigestion yields overhanging 3’-ends that can invade another DNA duplexRad50 and Mre11 collaborate to carry out this reaction2222.4 Gene ConversionWhen 2 similar, non-identical DNA sequences interact, possibility exists for gene conversionConversion of one DNA sequence into that of anotherSequences participating in gene conversions can be:Alleles, as in meiosisNonallelic genes, such as the MAT genes that determine mating type in yeast23Gene Conversion Model Strand exchange event with branch migration during sporulation has resolved to yield 2 duplex DNAs with patches of heteroduplex24Gene Conversion Without Mismatch RepairConsider from the middle of the DSB recombination schemeInvading strand is partially resectedDNA repair synthesis more extensiveBranch migration and resolution do not change nature of the 4 DNA strands25