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1、Presentation,Introduction of DNA RecombinationHaoran ZhangDepartment of Chemical and Biological EngineeringTufts University,DNA Recombination,RolesTypesExamples,Biological Roles for Recombination,Generating new gene/allele combinations(crossing over during meiosis)Generating new genes(e.g.,Immuno-gl
2、obulin rearrangement)Integration of a specific DNA elementDNA repair,Practical Uses of Recombination,1.Used to map genes on chromosomes 2.Making transgenic cells and organisms,Types of Recombination,Homologous-occurs between sequences that are nearly identical(e.g.,during meiosis)Site-Specific-occur
3、s between sequences with a limited stretch of similarity;involves specific sitesTransposition DNA element moves from one site to another,usually little sequence similarity involved,Examples of Recombination,Fig.22.1,Holliday ModelR.Holliday(1964)Holliday Junctions form during recombination HJs can b
4、e resolved 2 ways,patch,EM of a Holliday Junction w/a few melted base pairs around junction,Nonreciprocalrecombination,The recBCD Pathway of Homologous Recombination,3 steps of strand exchange:Pre-synapsis:recA coats single stranded DNA(accelerated by SSB,get more relaxed structure)Synapsis:alignmen
5、t of complementary sequences in SS and DS DNA3.Post-synapsis or strand-exchange:SS DNA replaces the same strand in the duplex to form a new DS DNA(requires ATP hydrolysis),The recBCD Pathway of Homologous Recombination Part I:Nicking and Exchanging,recBCD Pathway of Homologous Recombination Part I:N
6、icking and Exchanging,A nick is created in one strand by recBCD at a Chi sequence(GCTGGTGG),found every 5000 bp.Unwinding of DNA containing Chi sequence by recBCD allows binding of SSB and recA.recA promotes strand invasion into homologous DNA,displacing one strand.The displaced strand base-pairs wi
7、th the single strand left behind on the other chromosome.The displaced and now paired strand is nicked(by recBCD?)to complete strand exchange.,recBCD Pathway of Homologous RecombinationPart II:Branch Migration and Resolution,recBCD Pathway of Homologous Rec.Part II:Branch Migration and Resolution,Ni
8、cks are sealed Holliday JunctionBranch migration(ruvA+ruvB)Resolution of Holliday Junction(ruvC),RecBCD:A complex enzyme,RecBCD has:Endonuclease subunits(recBCD)that cut one DNA strand close to Chi sequence.DNA helicase activity(recBC subunit)andDNA-dependent ATPase activityunwinds DNA to generate S
9、S regions,RecA,38 kDa protein that polymerizes onto SS DNA 5-3Catalyzes strand exchange,also an ATPaseAlso binds DS DNA,but not as strongly as SS,RuvA and RuvB,DNA helicase that catalyzes branch migrationRuvA tetramer binds to HJ(each DNA helix between subunits)RuvB is a hexamer ring,has helicase&AT
10、Pase activity 2 copies of ruvB bind at the HJ(to ruvA and 2 of the DNA helices)Branch migration is in the direction of recA mediated strand-exchange,RuvB,RuvA,RuvC:resolvase,Endonuclease that cuts 2 strands of HJBinds to HJ as a dimer Consensus sequence:(A/T)TT(G/C)-occurs frequently in E.coli genome-branch migration needed to reach consensus sequence!,RuvC bound to Holliday junction,Thanks,
