基因的分子生物學RecombinationandRepair

1、會計學1基因的分子生物學基因的分子生物學RecombinationandRepair第1頁/共37頁2007-04-272Homologous recombination (generalized recombination) involves a reciprocal exchange of sequences of DNA, e.g. between two chromosomes that carry the same genetic loci.Site-specific recombination (Specialized recombination) occurs between 2

2、 specific sequences, as in phage integration/excision or resolution of cointegrate structures during transposition.Transposition refers to the movement of a transposon to a new site in the genome.Copy choice is a type of recombination used by RNA viruses, in which the RNA polymerase switches from on

3、e template to another during synthesis.Figure 15.1 Generalized recombination can occur at any point along the lengths of two homologous DNAs.Figure 15.2Figure Introduction第1頁/共37頁第2頁/共37頁2007-04-273A bivalent is the structure containing all four chromatids (two representing each homologue

4、) at the start of meiosis.Synapsis (聯(lián)會聯(lián)會) describes the association of the 2 pairs of sister chromatids (representing homologous chromosomes) that occurs at the start of meiosis; the resulting structure is called a bivalent.Chromosome pairing is the coupling of the homologous chromosomes at the star

5、t of meiosis.The synaptonemal complex describes the morphological structure of synapsed chromosomes.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 h

6、eteroduplex DNA around the site of joining).A chiasma (交叉交叉) (pl. chiasmata) is a site at which two homologous chromosomes appear to have exchanged material during meiosis.Figure 15.4 Recombination occurs during the first meiotic prophase. The stages of prophase are defined by the appearance of the

7、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.4.15.2 Homologous recombination occurs between synapsed chromos

8、omes第2頁/共37頁第3頁/共37頁2007-04-274Heteroduplex DNA (Hybrid DNA) is generated by base pairing between complementary single strands derived from the different parental duplex molecules; it occurs during genetic recombination.A Holliday structure is an intermediate structure in homologous recombination, w

9、here the two duplexes of DNA are connected by the genetic material exchanged between two of the four strands, one from each duplex. A joint molecule is said to be resolved when nicks in the structure restore two separate DNA duplexes.Splice recombinant DNA results from a Holliday junction being reso

10、lved by cutting the non-exchanged strands. Both strands of DNA before the exchange point come from one chromosome; the DNA after the exchange point come from the homologous chromosome.Patch recombinant DNA results from a Holliday junction being resolved by cutting the exchanged strands. The duplex i

11、s largely unchanged, except for a DNA sequence on one strand that came from the homologous chromosome.4.15.3 Breakage and reunion involves heteroduplex DNA第3頁/共37頁第4頁/共37頁2007-04-275Figure 15.5 Recombination between 2 paired duplex DNAs could involve reciprocal single-strand exchange, branch migrati

12、on, and nicking.Figure 15.6 Branch migration can occur in either direction when an unpaired single strand displaces a paired strand.Figure 15.7 Resolution of a Holliday junction can generate parental or recombinant duplexes, depending on which strands are nicked. Both types of product have a region

13、of heteroduplex DNA.第4頁/共37頁第5頁/共37頁2007-04-276A double-strand break (DSB) occurs when both strands of a DNA duplex are cleaved at the same site. Genetic recombination is initiated by double-strand breaks. The cell also has repair systems that act on double-strand breaks created at other times.Figur

14、e 15.8 Recombination is initiated by a double-strand break, followed by formation of single-stranded 3 ends, one of which migrates to a homologous duplex.4.15.4 Double-strand breaks initiate recombination第5頁/共37頁第6頁/共37頁2007-04-277The synaptonemal complex describes the morphological structure of syn

15、apsed chromosomes.An axial element is a proteinaceous structure around which the chromosomes condense at the start of synapsis.A lateral element is a structure in the synaptonemal complex. It is an axial element that is aligned with the axial elements of other chromosomes.The central element is a st

16、ructure that lies in the middle of the synaptonemal complex, along which the lateral elements of homologous chromosomes align. Recombination nodules (Node) are dense objects present on the synaptonemal complex; they may represent protein complexes involved in crossing-over.Figure 15.10 Each pair of

17、sister chromatids has an axis made of cohesins. Loops of chromatin project from the axis. The synaptonemal complex is formed by linking together the axes via zip proteins.4.15.5 Recombining chromosomes are connected by the synaptonemal complex第6頁/共37頁第7頁/共37頁2007-04-2784.15.6 The synaptonemal comple

18、x forms after double-strand breaksFigure 15.11 Spo11 is covalently joined to the 5 ends of double-strand breaks.Figure 15.12 Double-strand breaks appear when axial elements form, and disappear during the extension of synaptonemal complexes. Joint molecules appear and persist until DNA recombinants a

19、re detected at the end of pachytene (粗線期粗線期).第7頁/共37頁第8頁/共37頁2007-04-279 The RecBCD complex has nuclease and helicases activities. It binds to DNA downstream of a chi sequence, unwinds the duplex, and degrades one strand from 35 as it moves to the chi site. The chi site triggers loss of the RecD sub

20、unit and nuclease activity.Figure 15.13 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.4.15.8 The bacterial RecBCD system is stimulated by chi sequences第8頁/共37

21、頁第9頁/共37頁2007-04-2710Figure 15.14 RecA promotes the assimilation of invading single strands into duplex DNA so long as one of the reacting strands has a free end.Figure 15.15 RecA-mediated strand exchange between partially duplex and entirely duplex DNA generates a joint molecule with the same struc

22、ture as a recombination intermediate.Single-strand assimilation (Single-strand uptake) 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.4.15.9 Strand-transfer proteins cataly

23、ze single-strand assimilation (同化同化)第9頁/共37頁第10頁/共37頁2007-04-2711 The Ruv complex acts on recombinant junctions. RuvA recognizes the structure of the junction and RuvB is a helicase that catalyzes branch migration. RuvC cleaves junctions to generate recombination intermediates.Figure 15.17 Bacterial

24、 enzymes can catalyze all stages of recombination in the repair pathway following the production of suitable substrate DNA molecules.Figure 15.16 RuvAB is an asymmetric complex that promotes branch migration of a Holliday junction.4.15.10 The Ruv system resolves Holliday junctions第10頁/共37頁第11頁/共37頁2

25、007-04-2712Supercoiling describes the coiling of a closed duplex DNA in space so that it crosses over its own axis.Figure 15.20 Separation of the strands of a DNA double helix could be achieved in several ways.4.15.12 Supercoiling affects the structure of DNA第11頁/共37頁第12頁/共37頁2007-04-2713A type I to

26、poisomerase is an enzyme that changes the topology of DNA by nickingand resealing one strand of DNA.A type II topoisomerase is an enzyme that changes the topology of DNA by nickingand resealing both strands of DNA.Figure 15.21 The topological structure of DNA is changed during replication and transc

27、ription. Strand separation for replication (or transcription) requires a base turn of DNA to be unwound. Transcription creates positive supercoils ahead of the RNA polymerase. Replication of a circular template produces two catenated daughter templates.4.15.13 Topoisomerases relax or introduce super

28、coils in DNAType I enzymes act by breaking a single strand of DNA;type II enzymes act by making double-strand breaks.第12頁/共37頁第13頁/共37頁2007-04-2714Single-strand passage is a reaction catalyzed by type I topoisomerase in which one section of single-stranded DNA is passed through another strand.Figure

29、 15.22 Bacterial type I topoisomerases recognize partially unwound segments of DNA and pass one strand through a break made in the other.Figure 15.23 Type II topoisomerases can pass a duplex DNA through a double-strand break in another duplex.4.15.14 Topoisomerases break and reseal strands第13頁/共37頁第

30、14頁/共37頁2007-04-2715The sign inversion model describes the mechanism of DNA gyrase (促旋酶促旋酶). DNA gyrase binds a positive supercoil (inducing a compensatory negative supercoil elsewhere on the closed circular DNA), breaks both strands in one duplex, passes the other duplex through, and reseals the st

31、rands.Enzyme turnover is the process through which the enzyme returns to its original shape, enabling the enzyme to catalyze another reaction. E. coli gyrase is a type II topoisomerase that used hydrolysis of ATP to provide energy to introduce negative supercoils into DNA.Figure 15.24 DNA gyrase may

32、 introduce negative supercoils in duplex DNA by inverting a positive supercoil.4.15.15 Gyrase functions by coil inversion第14頁/共37頁第15頁/共37頁2007-04-2716Site-specific recombination (Specialized recombination) occurs between two specific sequences, as in phage integration/excision or resolution of coin

33、tegrate structures during transposition.Prophage is a phage genome covalently integrated as a linear part of the bacterial chromosome.Integration of viral or another DNA sequence describes its insertion into a host genome as a region covalently linked on either side to the host sequences.The excisio

34、n step in an excision-repair system consists of removing a single-stranded stretch of DNA by the action of a 5-3 exonuclease.A secondary attachment site is a locus on the bacterial chromosome into which phage lambda integrate inefficiently because the site resembles the att site.Figure 15.25 Circula

35、r 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.4.15.16 Specialized recombination involves specific sites第15頁/共37頁第16頁/共37頁2007-04-2717Figure 15.27 Staggered (交錯) cleavag

36、es in the common core sequence of attP and attB allow crosswise reunion to generate reciprocal recombinant junctions. 4.15.17 Site-specific recombination involves breakage and reunion第16頁/共37頁第17頁/共37頁2007-04-27184.15.18 Site-specific recombination resembles topoisomerase activityFigure 15.28 Integr

37、ases catalyze recombination by a mechanism similar to topoisomerases. Staggered cuts are made in DNA and the 3-phosphate end is covalently linked to a tyrosine in the enzyme. Then the free hydroxyl group of each strand attacks the P-Tyr link of the other strand. The first exchange shown in the figur

38、e generates a Holliday structure. The structure is resolved by repeating the process with the other pair of strands.Figure 15.29 A synapsed loxA recombination complex has a tetramer of Cre recombinases, with one enzyme monomer bound to each half site. Two of the four active sites are in use, acting

39、on complementary strands of the two DNA sites.第17頁/共37頁第18頁/共37頁2007-04-2719An intasome is a protein-DNA complex between the phage lambda integrase (Int)and the phage lambda attachment site (attP).Figure 15.30 Int and IHF bind to different sites in attP. The Int recognition sequences in the core reg

40、ion include the sites of cutting.Figure 15.31 Multiple copies of Int protein may organize attP into an intasome, which initiates site-specific recombination by recognizing attB on free DNA.4.15.19 Lambda recombination occurs in an intasome第18頁/共37頁第19頁/共37頁2007-04-2720A structural distortion is a ch

41、ange in the shape of a molecule.A pyrimidine dimer is formed when ultraviolet irradiation generates a covalent link directly between two adjacent pyrimidine bases in DNA. It blocks DNA replication.Photoreactivation uses a white-light-dependent enzyme to split cyclobutane pyrimidine dimers formed by

42、ultraviolet light.Mismatch repair corrects recently inserted bases that do not pair properly. The process preferentially corrects the sequence of the daughter strand by distinguishing the daughter strand and parental strand, sometimes on the basis of their states of methylation.Excision repair descr

43、ibes a type of repair system in which one strand of DNA is directly excised and then replaced by resynthesis using the complementary strand as template.Recombination-repair is a mode of filling a gap in one strand of duplex DNA by retrieving a homologous single strand from another duplex.Error-prone

44、 synthesis occurs when DNA incorporates noncomplementary bases into the daughter strand.4.15.20 Repair systems correct damage to DNA第19頁/共37頁第20頁/共37頁2007-04-2721Figure 15.32 Deamination of cytosine creates a U-G base pair. Uracil is preferentially removed from the mismatched pair.Figure 15.33 A rep

45、lication error creates a mismatched pair that may be corrected by replacing one base; if uncorrected, amutation is fixed in one daughter duplex.Figure 15.34 Ultraviolet irradiation causes dimer formation between adjacent thymines. The dimer blocks replication and transcription.Figure 15.35 Methylati

46、on of a base distorts the double helix and causes mispairing at replication.第20頁/共37頁第21頁/共37頁2007-04-2722Figure 15.36 Depurination removes a base from DNA, blocking replication and transcription.Figure 15.37 Repair genes can be classified into pathways that use different mechanisms to reverse or by

47、pass damage to DNA.Figure 15.38 Excision-repair directly replaces damaged DNA and then resynthesizes a replacement stretch for the damaged strand.第21頁/共37頁第22頁/共37頁2007-04-2723Incision (切開切開) is a step in a mismatch excision repair system. An endonuclease recognizes the damaged area in the DNA, and

48、isolates it by cutting the DNA strand on both sides of the damage.The excision(切除切除) step in an excision-repair system consists of removing a single-stranded stretch of DNA by the action of a 5-3 exonuclease. The Uvr system makes incisions 12 bases apart on both sides of damaged DNA, removes the DNA

49、 between them, and resynthesizes new DNA.Figure 15.39 Excision-repair removes and replaces a stretch of DNA that includes the damaged base(s).4.15.21 Excision repair systems in E. coli第22頁/共37頁第23頁/共37頁2007-04-2724 Uracil and alkylated bases are recognized by glycosylases and removed directly from D

50、NA. Pyrimidine dimers are reversed by breaking the covalent bonds between them. Methylases add a methyl group to cytosine. All these types of enzyme act by flipping the base out of the double helix, where, depending on the reaction, it is either removed or is modified and returned to the helix.Figur

51、e 15.41 A glycosylase removes a base from DNA by cleaving the bond to the deoxyribose.Figure 15.42 A glycosylase hydrolyzes the bond between base and deoxyribose (using H20), but a lyase takes the reaction further by opening the sugar ring (using NH2).Figure 15.43 A methylase flips the target cytosi

52、ne out of the double helix in order to modify it. 4.15.22 Base flipping is used by methylases and glycosylases第23頁/共37頁第24頁/共37頁2007-04-2725Figure 15.45 GATC sequences are targets for the Dam methylase after replication. During the period before this methylation occurs, the nonmethylated strand is t

53、he target for repair of mismatched bases.4.15.24 Controlling the direction of mismatch repair第24頁/共37頁第25頁/共37頁2007-04-2726Figure 15.46 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

54、mismatch site.Figure 15.47 The MutS/MutL system initiates repair of mismatches produced by replication slippage.第25頁/共37頁第26頁/共37頁2007-04-2727Single-strand exchange is a reaction in which one of the strands of a duplex of DNA leaves its former partner and instead pairs with the complementary strand

55、in another molecule, displacing its homologue in the second duplex.Figure 15.48 An E. coli retrieval system uses a normal strand of DNA to replace the gap left in a newly synthesized strand opposite a site of unrepaired damage.4.15.25 Recombination-repair systems in E. coli第26頁/共37頁第27頁/共37頁2007-04-

56、27284.15.26 Recombination is an important mechanism to recover from replication errorsFigure 15.49 A replication fork stalls when it reaches a damaged site in DNA. Branch migration occurs to move the fork backward, and the two daughter strands pair to form a duplex. After the damage has been repaire

57、d, a helicase may cause forward branch migration to restore the structure of the fork. Arrowheads indicate 3 ends.第27頁/共37頁第28頁/共37頁2007-04-2729Figure 15.50 The structure of a stalled replication fork resembles a Holliday junction and can be resolved in the same way by resolvases (溶解酶溶解酶). The resul

58、ts depend on whether the site of damage contains a nick. Result 1 shows that a double-strand break is generated by cutting a pair of strands at the junction. Result 2 shows a second DSB is generated at the site of damage if it contains a nick. Figure 15.51 When a replication fork stalls, recombinati

59、on-repair can place an undamaged strand opposite the damaged site. This allows replication to continue.第28頁/共37頁第29頁/共37頁2007-04-2730An SOS response in E. coli describes the coordinate induction of many enzymes, including repair activities, in response to irradiation or other damage to DNA; results

60、from activation of protease activity by RecA to cleave LexA repressor.The SOS box is the DNA sequence (operator) of 20 bp recognized by LexA repressor protein.Figure 15.52 The LexA protein represses many genes, including repair functions, recA and lexA. Activation of RecA leads to proteolytic cleava