分子生物學(xué)資料：chapter9 Homologous recombination-2

1、Chapter 9 Homologous Recombination at the Molecular Level DNA Breaks Are Common and Initiate Recombination, Models for Homologous Recombination, Homologous Recombination Protein Machines, Homologous Recombination in Eukaryotes, Mating-Type Switching, Genetic Consequences of the Mechanism of Homologo

2、us Recombination. RecA Protein Assembles on Single-Stranded DNA and Promotes Strand InvasionRecA is the central protein in homologous recombination. It is the founding member of a family of enzymes called strand-exchange proteins. These proteins catalyze the pairing of homologous DNA molecules. Pair

3、ing involves both the search for sequence matches between two molecules and the generation of regions of base pairing between these molecules.Figure 11-8Figure 11-9Figure 11-10Polarity of RecA assemblyFigure 11-11Newly Base-Paired Partners Are Established within the RecA FilamentRecA-catalyzed stran

4、d exchange can be divided into distinct reaction stages. First, the RecA filament must assemble on one of the participating DNA molecules. Assembly occurs on a molecule containing a region of ssDNA, such as an ssDNA tail. This RecAssDNA complex is the active form that participates in the search for

5、a homology. During this search, RecA must “l(fā)ook” for base-pair complementarity between the DNA within the filament and a new DNA molecule. This homology search is promoted by RecA because the filament structure has two distinct DNA-binding sites: a primary site (bound by the first DNA molecule) and

6、a secondary site. This secondary DNA-binding site can be occupied by double-stranded DNA. Binding to this site is rapid, weak, transient, andimportantlyindependent of DNA sequence. In this way, the RecA filament can bind and rapidly “sample” huge stretches of DNA for sequence homology.Figure 11-12Re

7、cA Homologs Are Present in All OrganismsStrand-exchange proteins of the RecA family are present in all forms of life. The best-characterized members are RecA from Eubacteria, RadA from Archaea, Rad51 and Dmc1 from Eukaryota, and the bacteriophage T4 UvsX protein. These proteins form filaments simila

8、r to those made by RecA and likely function in an analogous manner.Figure 11-16The RuvAB Complex Specifically Recognizes Holliday Junctions and Promotes Branch MigrationThe two recombining DNA molecules are connected by a DNA branch known as a Holliday junction. Movement of the site of this branch r

9、equires exchange of DNA base pairs between the two homologous DNA duplexes. RuvA protein is a Holliday junctionspecific DNA-binding protein that recognizes the structure of the DNA junction, regardless of its specific DNA sequence. RuvA recognizes and binds to Holliday junctions and recruits the Ruv

10、B protein to this site. RuvB is a hexameric ATPase. The RuvB ATPase provides the energy to drive the exchange of base pairs that moves the DNA branch. Figure 11-17RuvC Cleaves Specific DNA Strands at the Holliday Junction to Finish RecombinationResolution by RuvC occurs when RuvC recognizes the Holl

11、iday junctionlikely in a complex with RuvA and RuvBand specifically nicks two of the homologous DNA strands that have the same polarity. This cleavage results in DNA ends that terminate with 5-phosphates and 3-OH groups that can be directly joined by DNA ligase.Despite recognizing a structure rather

12、 than a specific sequence, RuvC cleaves DNA with modest sequence specificity. Cleavage takes place only at sites conforming to the consensus 5-A/T-T-T-G/C. Cleavage occurs after the second T in this sequence. Sequences with this consensus are found frequently in DNA, averaging once every 64 nucleoti

13、des. Figure 11-18HOMOLOGOUS RECOMBINATION IN EUKARYOTESHomologous Recombination Has Additional Functions in EukaryotesHomologous recombination is also required for DNA repair and the restarting of collapsed replication forks in eukaryotic cells.Most importantly, homologous recombination is critical

14、for meiosis. During meiosis, homologous recombination is required for proper chromosome pairing and thus for maintaining the integrity of the genome. This recombination also reshuffles genes between the parental chromosomes, ensuring variation in the sets of genes passed to the next generation.Figur

15、e 11-19Homologous Recombination Is Required for Chromosome Segregation during MeiosisFigure 11-20Programmed Generation of Double-Stranded DNA Breaks Occurs during MeiosisThe Spo11 protein cuts the DNA at many chromosomal locations, with little sequence selectivity, but at a very specific time during

16、 meiosis. Spo11-mediated DNA cleavage occurs right around the time when the replicated homologous chromosomes start to pair. Spo11 cut sites, although frequent, are not randomly distributed along the DNA. Rather, the cut sites are located most commonly in chromosomal regions that are not tightly pac

17、ked with nucleosomes, such as promoters controlling gene transcription. Regions of DNA that experience a high frequency of DSBs also show a high frequency of recombination. Thus, the most commonly used Spo11 DNA cleavage sites, like Chi sites, are hot spots for recombination.Figure 11-22Figure 11-23

18、MRX Protein Processes the Cleaved DNA Ends for Assembly of the RecA-Like Strand-Exchange ProteinsMRX is composed of protein subunits called Mre11, Rad50, and Xrs2. The MRX-dependent 5-to-3 resection generates the long ssDNA tails with 3 ends that are often 1 kb or longer. The MRX complex is also tho

19、ught to remove the DNA-linked Spo11.The strand-exchange proteins Dmc1 and Rad51 then assemble on the ssDNA tails. Both proteins participate in recombination, but how they work together is not known. Dmc1 Is a RecA-Like Protein That Specifically Functions in Meiotic Recombination Eukaryotes encode tw

20、o well-characterized homologs of the bacterial RecA protein: Rad51 and Dmc1. Both proteins function in meiotic recombination. Whereas Rad51 is widely expressed in cells dividing mitotically and meiotically, Dmc1 is expressed only as cells enter meiosis.Many Proteins Function Together to Promote Meio

21、tic RecombinationVarious other proteins have been shown to be involved with Rad51 to help promote recombination and DSB repair. Rad52 is another essential recombination protein that interacts with Rad51. Rad52 functions to promote assembly of Rad51 DNA filaments, the active form of Rad51. It does th

22、is by antagonizing the action of RPA, the major ssDNA-binding protein present in eukaryotic cells. Rad52 protein also promotes the annealing and base pairing of complementary ssDNA molecules, and this activity may also play a role in the strand-pairing reactions that occur during initiation of recom

23、bination. The product of the BRCA2 gene also participates in Rad51-mediated DSB repair.A complex containing a Rad51-like protein, called Rad51C, and a second protein, called XRCC3, has been found to contain Holliday junction resolvase activity. Similarly, members of a family of enzymesthe RecQ helic

24、asesplay critical roles in homologous recombination during DSB repair and are likely also involved in meiosis.Figure 11-24MATING-TYPE SWITCHINGIn addition to promoting DNA pairing, DNA repair, and genetic exchange, homologous recombination can also serve to change the DNA sequence at a specific chro

25、mosomal location. This type of recombination is sometimes used to regulate gene expression. For example, recombination controls the mating type of the budding yeast S. cerevisiae by switching which mating-type genes are present at a specific location that is being expressed in that organisms genome.

26、Haploid S. cerevisiae cells can be either of two mating types, a or . In addition, when an a cell and an cell come into close proximity, they can fuse (i.e., “mate”) to form an a/ diploid cell. The a/ cell may then go through meiosis to form two haploid a cells and two haploid cells.The mating-type

27、genes expressed in a given cell are those found at the mating-type locus (MAT locus) in that cell. Thus, in a cells, the a1 gene is present at the MAT locus, whereas in cells, the 1 and 2 genes are present at the MAT locus. Figure 19-21The mating-type genes encode transcriptional regulators. These r

28、egulators control expression of target genes whose products define each cell type. Thus, in a cells, the a1 gene is present at the MAT locus, whereas in cells, the 1 and 2 genes are present at the MAT locus.Figure 11-25MATING-TYPE SWITCHINGFigure 11-26aMating-Type Switching Is Initiated by a Site-Sp

29、ecific Double-Strand BreakMating-type switching is initiated by the introduction of a DSB at the MAT locus. This reaction is performed by a specialized DNA-cleaving enzyme, called the HO endonuclease.Figure 11-26b5-to-3 resection of the DNA at the site of the HO-induced break occurs by the same mech

30、anism used during meiotic recombination. Thus, resection depends on the MRXprotein complex and is specific for the strands that terminate with 5 ends. In contrast, the strands terminating with 3 ends are very stable and not subjected to nuclease digestion.Once the long 3 ssDNA tails have been genera

31、ted, this DNA becomes coated by the Rad51 and Rad52. These Rad51 proteincoated strands then search for homologous chromosomal regions to initiate strand invasion and genetic exchange.Figure 11-26cThe Rad51-coated 3 ssDNA tails from the MAT locus “choose” the DNA at either the HMR or HML locus for strand invasion. If the DNA sequence at MAT Is a, t