《分子生物学 Molecular Biology》课程教学课件（PPT讲稿，英文版）Chapter 22 Homologous Recombination

Chapter 22 Homologous Recombination @Rafferty,J.B.,S.E.Sedelnikova,D.Hargreaves,P.J.Artymink,P.J.Baker,G.J.Sharples, A.A.Mahdi,R.G.Lloyd,and D.W.Rice,Crystal structure of DNA recombination protein RuvA

Chapter 22 Homologous Recombination

homologous recombination:a crossover()event that joins DNA segments that were previously separated.Recombination can be intramolecular(分子内的)or intrermolecular. CopyrightThe McGraw-Hill Companies,Inc Permission required for reproduction or display Intermolecular: (a)Single crossover: (b)Double crossover Intramolecular: (a)Direct repeats: (b)Inverted repeats:

homologous recombination: a crossover(交换)event that joins DNA segments that were previously separated．Recombination can be intramolecular(分子内的) or intrermolecular

22.1 The RecBCD Pathway for Homologous Recombination(同源重组的RecBCD途径) The recombination process begins when the RecBCD protein,the product of the recB,-C and-D genes, binds to a DNA double-stranded break(DSB)and uses its DNA helicase activity to unwind the DNA toward a so-called chi site,x,which has the sequence 5'- GCTGGTGG-3'.Chi sites are found on average every 5000 bp in the E.coli genome. RecBCD also has double-stranded and single- stranded exonuclease and single-stranded endonuclease activities.These allow RecBCD to produce a single-stranded tail,which can then be coated by RecA protein

22.1 The RecBCD Pathway for Homologous Recombination(同源重组的RecBCD途径) ✓ The recombination process begins when the RecBCD protein，the product of the recB，-C and-D genes， binds to a DNA double-stranded break(DSB) and uses its DNA helicase activity to unwind the DNA toward a so-called chi site,χ，which has the sequence 5’- GCTGGTGG-3’. Chi sites are found on average every 5000 bp in the E.coli genome． ✓ RecBCD also has double-stranded and single￾stranded exonuclease and single-stranded endonuclease activities．These allow RecBCD to produce a single-stranded tail，which can then be coated by RecA protein．

5 ◆3 Chi a protruding end.cc (a)The RecBCD proteinbinds at a double- stranded DNA break,and the DNA helicase (b)Strand invasion:D-loop formation activity of RecBCD then unwinds the DNA cc toward a Chi site,ultimately creating a 3'- Scan for homology. ss DNA that is coated with RecA protein and SSB. (b)RecA promotes invasion of another DNA dy Nick duplex,forming a D loop. Strand exchange (RecA+SSB) (C)RecA helps the invading strand scan for a region of homology in the recipient DNA (e) Repair gaps and seal nicks (Holliday junction) duplex.Here,the invading strand has base- 3 paired with a homologous region,releasing Branch migration(RuvA RuvB) SSB and RecA. (g)Resolution (RuvC) ative resolution (RuvC)

(a)The RecBCD proteinbinds at a double￾stranded DNA break，and the DNA helicase activity of RecBCD then unwinds the DNA toward a Chi site，ultimately creating a 3’- ss DNA that is coated with RecA protein and SSB. (b)RecA promotes invasion of another DNA duplex, forming a D loop． (C)RecA helps the invading strand scan for a region of homology in the recipient DNA duplex．Here, the invading strand has base￾paired with a homologous region，releasing SSB and RecA

opyright The McGraw-Hl Compar ■5 3 (d)Once a homologous region is found,a nick Chi in the looped-out DNA appears,perhaps (a)RecBCD unwinds DNA and leaves 3'-protruding end,coated with RecA caused by RecBCD.This permits the tail of 0G00000003 the newly nicked DNA to base-pair with the (b) Strand invasion:D-loop formation single-stranded region in the other DNA, probably aided by RecA and SSB. (c) Scan for homology (e)The remaining gap are filled in and nicks are sealed by DNA ligase.yielding a four stranded complex with a Holliday junction. Nick Strand exchange(RecA+SSB) (f)Branch migration occurs,sponsored by RuvA and RuvB.Notice that the branch has Repair gaps and seal nicks migrated to the right. (Holliday junction) (g and h)Nicking by RuvC resolves the 3 ◆3 structure into two molecules,crossover 15 Branch migration (RuvA+RuvB) recombinants or heteroduplexesheteroduplex (异源双链核酸分子). (g)Resolution(RuvC)

(d)Once a homologous region is found，a nick in the looped-out DNA appears, perhaps caused by RecBCD．This permits the tail of the newly nicked DNA to base-pair with the single-stranded region in the other DNA， probably aided by RecA and SSB． (e)The remaining gap are filled in and nicks are sealed by DNA ligase．yielding a four stranded complex with a Holliday junction． (f) Branch migration occurs, sponsored by RuvA and RuvB．Notice that the branch has migrated to the right． (g and h) Nicking by RuvC resolves the structure into two molecules，crossover recombinants or heteroduplexesheteroduplex (异源双链核酸分子)．

How does RecBCD create the 3'-terminal single-stranded DNA tail that initiates strand invasion? depend on the relative ATP and Mg2+concentrations Copyright The McGraw-Hill Companies,Inc.Permission required for reproduction or display RecBCD (a)(ATP)>(Mg2+) (d)(Mg2+)>(ATP) 00000 3 3 Ch Chi

How does RecBCD create the 3’-terminal single-stranded DNA tail that initiates strand invasion? depend on the relative ATP and Mg2+ concentrations

ATP >Mg2+ RecBC Chi (a)(ATP)>(Mg2+) (a)RecBCD binds to a blunt DNA end and its helicase activity begins unwinding the DNA, Chi creating a looped 3'-terminal strand. (b) 5 (b)When RecBCD reaches a Chi site,it cleaves the 3'-terminal DNA strand just downstream of Chi,and loads RecA protein onto the looped out portion of the strand. (c)RecBCD continues to unwind the DNA, leaving a 3'-teminal,single-stranded DNA coated with RecA. 00000003 Chi

ATP >Mg2+ (b)When RecBCD reaches a Chi site，it cleaves the 3‘-terminal DNA strand just downstream of Chi，and loads RecA protein onto the looped out portion of the strand． (c)RecBCD continues to unwind the DNA， leaving a 3'-teminal，single-stranded DNA coated with RecA． (a)RecBCD binds to a blunt DNA end and its helicase activity begins unwinding the DNA, creating a looped 3'-terminal strand．

ATP >Mg2+ RecBCD (d)RecBCD binds to a blunt DNA end ch and unwinds the DNA,forming a (d)(Mg2+)>(ATP) loop in the 3'-terminal.single- stranded region,and degrading the DNA behind the loop. (e)When RecBCD reaches a Chi site,it loads RecA onto the looped-out portion of the strand.Simultaneously,RecBCD ceases to degrade the 3'-terminal strand, cuts the 5'-terminal strand,and activates a 5'3'exonuclease activity. (f)As RecBCD continues to unwind the DNA.its 5'3'exonuclease degrades the 5'-terminal strand.leaving a 3'- terminal single-stranded DNA coated with RecA. 3 Chi

(d)RecBCD binds to a blunt DNA end and unwinds the DNA，forming a loop in the 3’-terminal．single￾stranded region，and degrading the DNA behind the loop． (e) When RecBCD reaches a Chi site，it loads RecA onto the looped-out portion of the strand．Simultaneously, RecBCD ceases to degrade the 3’-terminal strand， cuts the 5’-terminal strand，and activates a 5’→3’ exonuclease activity． (f)As RecBCD continues to unwind the DNA．its 5’→3’exonuclease degrades the 5’-terminal strand．leaving a 3’- terminal single-stranded DNA coated with RecA． ATP >Mg2+

小结 √大肠杆菌中由重组蛋白BCD发起的同源重组起始于一双链 DNA被另一两条链已断裂的双链产生的单链DNA侵入。由重 组蛋白BCD介导的两种不同方式均会产生由重组蛋白A包裹 的游离3末端。 √由侵入链形成D环，接着D环链的降解将导致分支中间体的 形成。在中间体内，由分支迁移作用产生了霍利迪连接，使 同源染色体的两条DNA单链发生了片断的交换。最终，通过 在两条交换链上形成裂口而使霍利迪连接被打开。 √这样可以产生两条具有相应补丁的非交换异源重组DNA,或 是两条已经交换了侧翼序列的交换重组DNA。侵入链上包裹 有重组蛋白A和单链结合蛋白，而且重组蛋白A可以使侵入 链和其互补链之间较易形成碱基互补配对。 √由于RuVA和RuVB具有解旋酶活性，这就使分支迁移可以进 行下去。RVC在霍利迪连接中形成裂口从而打开该连接形 成重组DNA

小结 ✓ 大肠杆菌中由重组蛋白BCD发起的同源重组起始于一双链 DNA被另一两条链已断裂的双链产生的单链DNA侵入。由重 组蛋白BCD介导的两种不同方式均会产生由重组蛋白A包裹 的游离3末端。 ✓ 由侵入链形成D环，接着D环链的降解将导致分支中间体的 形成。在中间体内，由分支迁移作用产生了霍利迪连接，使 同源染色体的两条DNA单链发生了片断的交换。最终，通过 在两条交换链上形成裂口而使霍利迪连接被打开。 ✓ 这样可以产生两条具有相应补丁的非交换异源重组DNA，或 是两条已经交换了侧翼序列的交换重组DNA。侵入链上包裹 有重组蛋白A和单链结合蛋白，而且重组蛋白A可以使侵入 链和其互补链之间较易形成碱基互补配对。 ✓ 由于RuvA和RuvB具有解旋酶活性，这就使分支迁移可以进 行下去。RuvC在霍利迪连接中形成裂口从而打开该连接形 成重组DNA

22.3 Meiotic Recombination meiosis in most eukaryotes is accompanied by recombination.This process shares many characteristics in common with homologous recombination in bacteria. In this section,we will examine the mechanism of meiotic recombination in yeast,with special emphasis on the initiating event,which is quite different from the models of initiation of bacterial homologous recombination presented earlier in this chapter

22.3 Meiotic Recombination meiosis in most eukaryotes is accompanied by recombination. This process shares many characteristics in common with homologous recombination in bacteria. In this section, we will examine the mechanism of meiotic recombination in yeast, with special emphasis on the initiating event, which is quite different from the models of initiation of bacterial homologous recombination presented earlier in this chapter