《分子生物学》5cha.ppt

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1、Chapter 10Homologous recombination Homologous recombination at the molecular levelat the molecular leveln nAll DNA is recombinant DNA.n nGenetic exchange works constantly to blend and rearrange chromosomes,most obviously during meiosis,when homologous chromosomes pair prior to the first nuclear divi

2、sion.n nDuring this pairing,genetic exchange between the chromosomes occurs.This exchange,classically termed crossing over,is one of the results of homologous recombination.1.1.Models for Homologous Recombination 2.2.Homologous Recombination Protein Machines3.3.Homologous Recombination in Eukaryotes

3、 4.4.Mating-Type Switching in prokaryotesAlignment:Breaks:Strand invasionHolliday junctionBranch migration:Movement of the Holliday junctionResolution:Cleavage of the Holliday junctionModels for homologous recombination1.The Holliday Model 2.The Double-Strand Break Repair Modeln nDouble-straned brea

4、ks in DNA arise quite frequently n nIn bacteria,the major biological role of homologous recombination is to repair DSBsn nIn addition to repairing DSBs in chromosomal DNA,homologous recombination promotes genetic exchange in bacterian nIn eukaryotic cells,homologous recombination is critical for rep

5、airing DNA Double stranded DNA breaks arise by numerous means and initiate homologous recombinationDamage in the DNA template can lead To DSB formation during DNA replicationHomologous recombination protein machinesHomologous recombination in prokaryotesl lThe RecBCD helicase/nuclease processes brok

6、en DNA molecules for recombination l lRecA protein assembles on single-stranded DNA and promotes strand invasionl lNewly base-paired partners are established within the RecA filamentl lRuvAB complex specifically recognizes Holliday junctions and promotes branch migrationl lRuvC cleaves specific DNA

7、strands at the Holliday junction to finish recombinationSteps of DNA processing by RecBCDPolar action of chin nRecA Protein:strand-exchange proteinn nThe active form of recA is a protein-DNA filament many subunits+many strands n nCoated with RecA make DNA highly extendedn nPolarity of RecA assemblyR

8、ecA Protein Assembles on Single-stranded DNA and Promotes Strand Invasion RecA Stabilises single stranded DNA RecA prevents secondary structure within the single stranded DNA RecA forces the single stranded DNA to take on a helical shape,effectively lowering the energy barrier to recombinationRecA P

9、rotein Assembles on Single-stranded DNA and Promotes Strand Invasion Polarity of RecA assemblyNewly base-paired partners are established within the RecA filamentn ncross-section of single DNA strand bound to RecA proteinn nHomology search is promoted by RecA.DNA in secondary site is testes for compl

10、ementarityn nJoint molecular:Base-paring between strands is switchedn nRecA is prone to bind the DNA products after strand exchange.RecA-catalyzed strand exchange can be divided into RecA-catalyzed strand exchange can be divided into different stagesdifferent stagesModel of two steps in search for h

11、omology and DNA strand exchange within the RecA filament Homologous recombination in prokaryotesl lThe RecBCD helicase/nuclease processes broken DNA molecules for recombination l lRecA protein assembles on single-stranded DNA and promotes strand invasionl lNewly base-paired partners are established

12、within the RecA filamentl lRuvAB complex specifically recognizes Holliday junctions and promotes branch migrationl lRuvC cleaves specific DNA strands at the Holliday junction to finish recombinationRuvAB complex specifically recognizes Holliday junctions and promotes branch migrationRuvA protein is

13、a Holliday junction specific DNA-binging protein that recognizes the structure of the DNA junctionRuvB is a hexametric ATPase:drive the exchange of base pair that move the DNA branchHigh resolution structure of the RuvA-DNA complex and High resolution structure of the RuvA-DNA complex and schematic

14、model of the RuvAB complex bound to Holliday schematic model of the RuvAB complex bound to Holliday junction DNAjunction DNARuvC cleaves specific DNA strands at the Holliday junction to finish recombinationCompletion of recombination requires that the Holliday Completion of recombination requires th

15、at the Holliday junction between the two recombining DNA molecules be junction between the two recombining DNA molecules be resolved resolved RuvCRuvC specifically nicks two of the homologous DNA specifically nicks two of the homologous DNA strands that have the same polarity strands that have the s

16、ame polarity RuvCRuvC recognize the structure rather than a special recognize the structure rather than a special sequence,but still with modest sequence,but still with modest sequence specificitysequence specificity High resolution structure of the RuvC resolves and schematic model of the RuvC dime

17、r bound to Holliday junction DNA1.1.Models for Homologous Recombination 2.2.Homologous Recombination Protein Machines3.3.Homologous Recombination in Eukaryotes 4.4.Mating-Type Switching 5.5.Genetic Consequences of the Mechanism of Homologous Recombinationin prokaryotesHomologous recombination has ad

18、ditional functions in eukaryotes Homologous recombination can repair double stranded breaks in DNA,to restart collapsed replication forks,and to allow a cells chromosomal DNA to recombine with DNA that enters via phage infection or junctionDuring meiosis,homologous recombination is required for prop

19、er chromosome pairing Recombination must be complete before the first nuclear division to allow the homologs to properly align and then separate The improper segregation of chromosomes,called nondisjunction,leads to a large number of gametes without the correct chromosome complementThe homologous re

20、combination events that occur during meiosis are called meiotic recombinationHomologous recombination is required for chromosome segregation during meiosisDNA dynamics during meiosisHomologous recombination in eukaryotes 1.1.Double stranded DNA breaks 2.2.MRX protein:RecBCD-like protein 3.3.Dmc1:Rec

21、A-like protein 4.4.Many protein together:Rad51 Rad52Mechanism of cleavage by spo11Overview of meiotic Overview of meiotic recombination pathwayrecombination pathwayDmc1-dependent recombination occurs preferentially between nonsister homologous chromatidsMany protein function together to promote meio

22、tic recombinationSome large protein-DNA complexes,known as recombination factoriesRad51,Dmc1,Rad521.1.Models for Homologous Recombination 2.2.Homologous Recombination Protein Machines3.3.Homologous Recombination in Eukaryotes 4.4.Mating-Type Switching 5.5.Genetic Consequences of the Mechanism of Homologous Recombinationin prokaryotesGenetic loci encoding mating-type information1.1.Models for Homologous Recombination 2.2.Homologous Recombination Protein Machines3.3.Homologous Recombination in Eukaryotes 4.4.Mating-Type Switching in prokaryotes