分子生物学 第十四章 重组和修复（Recombination and repair）

1、Chapter 14,Recombination and repair,14.1 Introduction14.2 Breakage and reunion involves heteroduplex DNA 14.3 Double-strand breaks initiate recombination 14.4 Double-strand breaks initiate snapsis14.5 Bacterial recombination involves single-strand assimilation14.6 Gene conversion accounts for intera

2、llelic recombination 14.7 Topological manipulation of DNA14.8 Specialized recombination involves breakage and reunion at specific sites 14.9 Repair systems correct damage to DNA 14.10 Excision repair systems in E. coli 14.11 Controlling the direction of mismatch repair 14.12 Retrieval systems in E.

3、coli 14.13 RecA triggers the SOS system 14.14 Eukaryotic repair systems,Bivalent is the structure containing all four chromatids (two representing each homologue) at the start of meiosis.Breakage and reunion describes the mode of genetic recombination, in which two DNA duplex molecules are broken at

4、 corresponding points and then rejoined crosswise (involving formation of a length of heteroduplex DNA around the site of joining).Site-specific recombination occurs between two specific (not necessarily homologous) sequences, as in phage integration/excision or resolution of cointegrate structures

5、during transposition.Synapsis describes the association of the two pairs of sister chromatids representing homologous chromosomes that occurs at the start of meiosis; resulting structure is called a bivalent.Synaptonemal complex describes the morphological structure of synapsed chromosomes.Transposi

6、tion refers to the movement of a transposon to a new site in the genome.,14.1 Introduction,Figure 14.1 Recombination occurs during the first meiotic prophase. The stages of prophase are defined by the appearance of the chromosomes, each of which consists of two replicas (sister chromatids), although

7、 the duplicated state becomes visible only at the end. The molecular interactions of any individual crossing-over event involve two of the four duplex DNAs.,14.1 Introduction,Figure 1.22 The ABO blood group locus codes for a galactosyltransferase whose specificity determines the blood group.,14.1 In

8、troduction,Branch migration describes the ability of a DNA strand partially paired with its complement in a duplex to extend its pairing by displacing the resident strand with which it is homologous.Hybrid DNA is another term for heteroduplex DNA.Recombinant joint is the point at which two recombini

9、ng molecules of duplex DNA are connected (the edge of the heteroduplex region).,14.2 Breakage and reunion involves heteroduplex DNA,Figure 14.1 Recombination occurs during the first meiotic prophase. The stages of prophase are defined by the appearance of the chromosomes, each of which consists of t

10、wo replicas (sister chromatids), although the duplicated state becomes visible only at the end. The molecular interactions of any individual crossing-over event involve two of the four duplex DNAs.,14.2 Breakage and reunion involves heteroduplex DNA,Figure 14.2 Recombination between two paired duple

11、x DNAs could involve reciprocal single-strand exchange, branch migration, and nicking.,14.2 Breakage and reunion involves heteroduplex DNA,Figure 14.3 Branch migration can occur in either direction when an unpaired single strand displaces a paired strand.,14.2 Breakage and reunion involves heterodup

12、lex DNA,Figure 14.4 Resolution of a Holliday junction can generate parental or recombinant duplexes, depending on which strands are nicked. Both types of product have a region of heteroduplex DNA.,14.2 Breakage and reunion involves heteroduplex DNA,Figure 14.5 Recombination is initiated by a double-

13、strand break, followed by formation of single-stranded 3 ends, one of which migrates to a homologous duplex.,14.3 Double-strand breaks initiate recombination,Figure 14.6 The synaptonemal complex brings chromosomes into juxtaposition. This example of Neotellia was kindly provided by M. Westergaard an

14、d D. Von Wettstein.,14.4 Double-strand breaks initiate synapsis,Figure 14.7 Double-strand breaks appear when axial elements form, and disappear during the extension of synaptonemal complexes. Joint molecules appear and persist until DNA recombinants are detected at the end of pachytene.,14.4 Double-

15、strand breaks initiate synapsis,Figure 14.8 Spo11 is covalently joined to the 5 ends of double-strand breaks.,14.4 Double-strand breaks initiate synapsis,Figure 14.9 RecBCD nuclease approaches a chi sequence from one side, degrading DNA as it proceeds; at the chi site, it makes an endonucleolytic cu

16、t, loses RecD, and retains only the helicase activity.,14.5 The bacterial RecBCD system is stimulated by chi sequences,Paranemic joint describes a region in which two complementary sequences of DNA are associated side by side instead of being intertwined in a double helical structure.Single-strand a

17、ssimilation describes the ability of RecA protein to cause a single strand of DNA to displace its homologous strand in a duplex; that is, the single strand is assimilated into the duplex.,14.6 RecA catalyzes single-strand assimilation,Figure 14.10 RecA promotes the assimilation of invading single st

18、rands into duplex DNA so long as one of the reacting strands has a free end.,14.6 RecA catalyzes single-strand assimilation,Figure 14.2 Recombination between two paired duplex DNAs could involve reciprocal single-strand exchange, branch migration, and nicking.,14.6 RecA catalyzes single-strand assim

19、ilation,Figure 14.11 RecA-mediated strand exchange between partially duplex and entirely duplex DNA generates a joint molecule with the same structure as a recombination intermediate.,14.6 RecA catalyzes single-strand assimilation,Figure 14.12 RuvAB is an asymmetric complex that promotes branch migr

20、ation of a Holliday junction.,14.6 RecA catalyzes single-strand assimilation,Figure 14.13 Bacterial enzymes can catalyze all stages of recombination in the repair pathway following the production of suitable substrate DNA molecules.,14.6 RecA catalyzes single-strand assimilation,Gene conversion is t

21、he alteration of one strand of a heteroduplex DNA to make it complementary with the other strand at any position(s) where there were mispaired bases.Postmeiotic segregation describes the segregation of two strands of a duplex DNA that bear different information (created by heteroduplex formation dur

22、ing meiosis) when a subsequent replication allows the strands to separate.,14.8 Gene conversion accounts for interallelic recombination,Figure 14.14 Spore formation in the Ascomycetes allows determination of the genetic constitution of each of the DNA strands involved in meiosis.,14.8 Gene conversio

23、n accounts for interallelic recombination,Supercoiling describes the coiling of a closed duplex DNA in space so that it crosses over its own axis.Topological isomers are molecules of DNA that are identical except for a difference in linking number.Twisting number of a DNA is the number of base pairs

24、 divided by the number of base pairs per turn of the double helix.Writhing number is the number of times a duplex axis crosses over itself in space.,14.9 Topological manipulation of DNA,Figure 14.14 Spore formation in the Ascomycetes allows determination of the genetic constitution of each of the DN

25、A strands involved in meiosis.,14.9 Topological manipulation of DNA,Figure 14.15 Separation of the strands of a DNA double helix could be achieved by several means.,14.9 Topological manipulation of DNA,Figure 9.18 E. coli sigma factors recognize promoters with different consensus sequences. (Numbers

26、 in the name of a factor indicate its mass.),14.9 Topological manipulation of DNA,Figure 14.16 Bacterial type I topoisomerases recognize partially unwound segments of DNA and pass one strand through a break made in the other.,14.9 Topological manipulation of DNA,Figure 14.17 Type II topoisomerases c

27、an pass a duplex DNA through a double-strand break in another duplex.,14.9 Topological manipulation of DNA,Figure 14.18 DNA gyrase may introduce negative supercoils in duplex DNA by inverting a positive supercoil.,14.9 Topological manipulation of DNA,att sites are the loci on a phage and the bacteri

28、al chromosome at which recombination integrates the phage into, or excises it from, the bacterial chromosome.,14.10 Specialized recombination involves breakage and reunion at specific sites,Figure 14.19 Circular phage DNA is converted to an integrated prophage by a reciprocal recombination between a

29、ttP and attB; the prophage is excised by reciprocal recombination between attL and attR.,14.10 Specialized recombination involves breakage and reunion at specific sites,Figure 14.20 Does recombination between attP and attB proceed by sequential exchange or concerted cutting?,14.10 Specialized recomb

30、ination involves breakage and reunion at specific sites,Figure 14.21 Staggered cleavages in the common core sequence of attP and attB allow crosswise reunion to generate reciprocal recombinant junctions.,14.10 Specialized recombination involves breakage and reunion at specific sites,Figure 14.2 Reco

31、mbination between two paired duplex DNAs could involve reciprocal single-strand exchange, branch migration, and nicking.,14.10 Specialized recombination involves breakage and reunion at specific sites,Figure 14.22 Int and IHF bind to different sites in attP. The Int recognition sequences in the core

32、 region include the sites of cutting.,14.10 Specialized recombination involves breakage and reunion at specific sites,Figure 14.23 The Int binding sites in the core lie on one face of DNA. The large circles indicate positions at which methylation is influenced by Int binding; the large arrows indica

33、te the sites of cutting. Photograph kindly provided by A. Landy.,14.10 Specialized recombination involves breakage and reunion at specific sites,Figure 14.24 Multiple copies of Int protein may organize attP into an intasome, which initiates site-specific recombination by recognizing attB on free DNA

34、.,14.10 Specialized recombination involves breakage and reunion at specific sites,Figure 14.25 Substitutions of individual bases create mismatched pairs that may be corrected by replacing one base; if uncorrected they cause a mutation in one daughter duplex.,14.11 Repair systems correct damage to DN

35、A,Figure 14.26 Modifications or removal of bases may cause structural defects that prevent replication or induce mutations in each replication cycle until they are removed.,14.11 Repair systems correct damage to DNA,Figure 14.14 Spore formation in the Ascomycetes allows determination of the genetic

36、constitution of each of the DNA strands involved in meiosis.,14.11 Repair systems correct damage to DNA,Excision of phage or episome or other sequence describes its release from the host chromosome as an autonomous DNA molecule.,14.12 Excision repair systems in E. coli,Figure 14.27 Excision-repair r

37、emoves and replaces a stretch of DNA that includes the damaged base(s).,14.12 Excision repair systems in E. coli,Figure 14.28 The Uvr system operates in stages in which UvrAB recognizes damage, UvrBC nicks the DNA, and UvrD unwinds the marked region.,14.12 Excision repair systems in E. coli,Figure 1

38、4.28 The Uvr system operates in stages in which UvrAB recognizes damage, UvrBC nicks the DNA, and UvrD unwinds the marked region.,14.12 Excision repair systems in E. coli,Figure 14.37 A helicase unwinds DNA at a damaged site, endonucleases cut on either side of the lesion, and new DNA is synthesized

39、 to replace the excised stretch.,14.12 Excision repair systems in E. coli,Figure 14.29 A glycosylase removes a base from DNA by cleaving the bond to the deoxyribose.,14.13 Base flipping is used by methylases and glycosylases,Figure 14.30 A glycosylase hydrolyzes the bond between base and deoxyribose

40、 (using H20), but a lyase takes the reaction further by pening the sugar ring (using NH2).,14.13 Base flipping is used by methylases and glycosylases,Figure 14.31 A methylase flips the target cytosine out of the double helix in order to modify it. Photograph kindly provided by Rich Roberts.,14.13 Ba

41、se flipping is used by methylases and glycosylases,Figure 14.32 Preferential removal of bases in pairs that have oxidized guanine is designed to minimize mutations.,14.15 Controlling the direction of mismatch repair,Figure 13.30 Replication of methylated DNA gives hemimethylated DNA, which maintains its state at GATC sites until the Dam methylase restores the fully methylated condition.,14.1