第31章 DNA的重组 DNA分子内或分子间遗传信息的重新组合 重组的形式多种多样： 真核生物减数分裂时染色体的交换 重组是进化的动力 遗传多样性的基础 将有利突变固定遗传

Recombination Homologous recombination Site-specific recombination Transposition Mutation Relevance An important reason for variable DNA sequences among different populations of the same species

1.同源重组：一般性重组，最基本的重组方式 两条同源DNA分子经过配对、断裂和再连接，而产生片段交换 主要发生在减数分裂时期染色体交叉，四分体形成 重点：Holiday中间体 过程：断裂、交叉、分支移动、弯曲、旋转、切开 2.位点特异性重组 3.转座重组

Homologous recombination ( 同源重组） The exchange of homologous regions between two DNA moleculs Diploid eukaryotes: crossing over Haploid prokaryotes: recA-dependent, Holliday model DNA repair in replication fork

Homologous Recombination occur between Homo-chromosome / Homo-seq. sister & non-sister chromatids transformation, transduction conjugation, transfection… large fragment exchange Recombination site is in hotspot mostly Recombinase be needed (RecA, BCD)

Rec A蛋白：促进单链同化及SOS反应，最重要的重组蛋白 Rec A蛋白结合在单链上，与同源双链交换，置换出另一条单链 同源重组可产生异源双链和同源双链 重组十分精确 两DNA分子具有75bp以上的同源区 并不要求完全同源 重组与复制、重组修复密切相关 需要多种酶的参与 Rec A蛋白：促进单链同化及SOS反应，最重要的重组蛋白 Rec A蛋白结合在单链上，与同源双链交换，置换出另一条单链 Rec BCD蛋白：产生参与重组的DNA单链 具有解链酶活性（ATPase）& 核酸外切酶活性

Rec BCD蛋白:三种酶活，移到chi位点时，切成具有游离3，端的单链，GCTGGTGG Ruv A 和Ruv B促进异源双链的形成 Ruv C拆分Holiday中间体 细菌的基因转移与重组： 接合： 性因子，F质粒 转化： 感受态 转导： 噬菌体为媒介 融合：

使crossover point 沿D.S.DNA 解旋的方向移端(migration) 使heteroduplex region 重新形成螺旋 使D.S. DNA nick release S.S. DNA(被RecA-p结合) RecBCD-p特异识别GCTGGTGGT 序列 并在其下游4-6bp处切断S.S.DNA χ(chi)—sequence 交换热点

Diploid eukaryotes: crossing over Homologous chromosomes line up in meiosis (when) The nonsister chromatids exchange equivalent sections (what)

Holliday Intermediate structure

Haploid prokaryotes recombination Between the two homologous DNA duplex (where) between the replicated portions of a partially duplicated DNA between the chromosomal DNA and acquired “foreign” DNA Holliday model (How)

recA-dependent bacterial homologous recombination Homologous DNA pairs 2. Nicks made near Chi (GCTGGTGG) sites by a nuclease. 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3. ssDNA carrying the 5’ ends of the nicks is coated by RecA to form RecA-ssDNA dilaments.

3. RecA-ssDNA filaments search the opposite DNA duplex for corresponding sequence (invasion). 4. form a four-branched Holliday structure 5. Branch migration

6. Resolving Holliday junction

RuvAB is an asymmetric complex that promotes branch migration of a Holliday junction.

Recombination based DNA repair at replication fork a. Replication encounter a DNA lesion b. Skip the lesion & re-initiate on the side of the lesion c. Fill the daughter strand gap by replacing it with the corresponding section from the parental sister strand d. post-replication repair of the left lesion

Rec.A-p Hydrogen bond reform heterodulpex

具有物种，基因和genome seq.的特异性 χ(chi)—sequence 具有物种，基因和genome seq.的特异性 in E.coli genome 1000 chi-seq. chi RecBCD DNA解旋 再螺旋 4-6bp OH RecA-p

2.位点特异性重组 重组发生在特异的重组位点上 可以是分子内，也可以是分子间 都是四条单链 同一DNA分子同向：切除 同一DNA分子反向：倒位 不同DNA分子：整合 Ig的基因重排

Site-specific recombination ( 位点专一重组） Exchange of non-homologous but specific pieces of DNA (what) Mediated by proteins that recognize specific DNA sequences. (how)

Specific Recombination Site-Dependent Specific Recombination Integrase (Int-ase be needed & not need RecA-p ) Conservative recombination

Site-specific recombination: bacteriophage l insertion l-encoded integrase (Int): makes staggered cuts in the specific sites Int and IHF (integration host factor encoded by bacteria): recombination and insertion l-encoded excisionase (XIS): excision of the phage DNA

p O p’ attP of λ attB of E.coli B O B’ B O P’ P O B’ Int (Integrase) IHF (Integration host factor) Xis (Excisionase) FIS (factor of inversion stimulation) B O P’ P O B’

Site-specific recombination: Antibody diversity H and L are all encoded by three gene segments: V, D, J V D J Two heavy chains (H) 250 15 5 Two light chains (L) 4 Enormous number (>108) of different H and L gene sequences can be produced by such a recombination

3.转座重组 转座因子：可以从染色体的一个位置移到另一个位置 插入序列 转座子 非复制转座与复制转座 都具有反向重复的序列

Transposition ( 转座作用） Requires no homology between sequences nor site-specific Relatively inefficient Require Transposase encoded by the transposon ( 转座 子）

Various transposons: In E. coli: IS elements/insertion sequence, 1-2 kb, comprise a transposase gene flanked by a short inverted terminal repeats Tn transposon series carry transposition elements and b-lactamase (penicillin resistance) Eukaryotic transposons, many are retrotransposons: Yeast Ty element encodes protein similar to RT (reverse transcriptase)

Simplified Transposition process

Transposition Recombination (replication recombination) Specific tansposable genetic element Independent on homo-sequence between Tn & target site Transpotase be needed Leads to insertion, deletion, inversion, rearrangement…