Chapter 14

Chapter Chapter 1414

Recombination Recombination and repairand 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 interallelic 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. 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 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 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.Transposition 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 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 Introduction

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 recombining 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 two 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 duplex DNAs could involve reciprocal single-strand exchange, branch migration, and nicking.

14.2 Breakage


heteroduplex DNA

Figure 14.3 Branch migration can occur in either direction when an unpaired single strand displaces a paired strand.

14.2 Breakage


heteroduplex 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


heteroduplex DNA

Figure 14.5 Recombination is initiated by a double-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 and 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-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 cut, 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 assimilation 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 strands into duplex DNA so long as one of the reacting strands has a free end.



14.6 RecA catalyzes single-

strand assimilation

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 migration of a Holliday junction.


Figure 14.13 Bacterial enzymes can catalyze all stages of recombination in the repair pathway following the production of suitable substrate DNA molecules.


Gene conversion is the 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 during 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 conversion 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 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

DNA strands involved in

meiosis.


Figure 14.15 Separation of the strands of a DNA double helix could be achieved by several means.


Figure 9.18 E. coli sigma factors recognize promoters with different consensus sequences. (Numbers in the name of a factor indicate its mass.)


Figure 14.16 Bacterial type I topoisomerases recognize partially unwound segments of DNA and pass one strand through a break made in the other.


Figure 14.17 Type II topoisomerases can pass a duplex DNA through a double-strand break in another duplex.


Figure 14.18 DNA gyrase may introduce negative supercoils in duplex DNA by inverting a positive supercoil.


att sites are the loci on a phage and the bacterial 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 attP 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?


Figure 14.21 Staggered cleavages in the common core sequence of attP and attB allow crosswise reunion to generate reciprocal recombinant junctions.


Figure 14.22 Int and IHF bind to different sites in attP. The Int recognition sequences in the core region include the sites of cutting.


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 indicate the sites of cutting. Photograph kindly provided by A. Landy.


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.


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 DNA

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.


Figure 14.14 Spore formation in the Ascomycetes allows determination of the genetic constitution of each of the DNA strands involved in meiosis.


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 removes and replaces a stretch of DNA that includes the damaged base(s).


Figure 14.28 The Uvr system operates in stages in which UvrAB recognizes damage, UvrBC nicks the DNA, and UvrD unwinds the marked region.


Figure 14.37 A helicase unwinds DNA at a damaged site, endonucleases cut on either side of the lesion, and new DNA is synthesized to replace the excised stretch.


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 (using H20), but a lyase takes the reaction further by pening the sugar ring (using NH2).


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.


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.


Figure 14.33 GATC sequences are targets for the Dam methylase after replication. During the period before this methylation occurs, the nonmethylated strand is the target for repair of mismatched bases.


Figure 14.34 MutS recognizes a mismatch and translocates to a GATC site. MutH cleaves the unmethylated strand at the GATC. Endonucleases degrade the strand from the GATC to the mismatch site.


Figure 14.31 An E. coli retrieval system uses a normal strand of DNA replace the gap left in a newly synthesized strand opposite a site of unrepaired damage.

14.16 Retrival systems in E. coli

Figure 14.32 The LexA protein represses many genes, including repair functions, recA and lexA,. Activation of RecA leads to proteolytic cleavage of LexA and induces all of these genes.

14.17 RecA triggers the SOS system

Figure 14.37 A helicase unwinds DNA at a damaged site, endonucleases cut on either side of the lesion, and new DNA is synthesized to replace the excised stretch.

14.18 Eukaryotic repair systems

Summary

Chapter 14

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