第 31 章 dna 的重组

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

原核生物不同亲代两组 DNA 遗传重组

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

Homologous recombination Site-specific recombination Transposition

RecombinationRecombination

Mutation RelevanceAn 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）

Diploid eukaryotes: crossing overHaploid prokaryotes: recA-dependent,

Holliday modelDNA repair in replication fork

The exchange of homologous regions between two DNA moleculs

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)

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：

• RecBCD-p 特异识别 GCTGGTGGT 序列 并在其下游 4-6bp 处切断 S.S.DNA

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 )

χ(chi)—sequence 交换热点

Diploid eukaryotes: crossing over1. Homologous chromosomes line up in meiosis (when)

2. The nonsister chromatids exchange equivalent sections (what)

Holliday Intermediate structure

Haploid prokaryotes recombination

Between the two homologous DNA duplex (where)

1. between the replicated portions of a partially duplicated DNA

2. between the chromosomal DNA and acquired “foreign” DNA

Holliday model (How)

2. Nicks made near Chi (GCTGGTGG) sites by a nuclease.

3. ssDNA carrying the 5’ ends of the nicks is coated by RecA to form RecA-ssDNA dilaments.

recA-dependent bacterial homologous recombination

1. Homologous DNA pairs

3’ 5’

3’ 5’

3’

3’

5’

5’

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

χ(chi)—sequence 具有物种，基因和 genome seq. 的特异性

in E.coli genome 1000 chi-seq.

chiRecBCD

4-6bpOH

RecA-p

DNA 解旋 再螺旋

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）

1. Exchange of non-homologous but specific pieces of DNA (what)

2. Mediated by proteins that recognize specific DNA sequences. (how)

Site-Dependent

Specific Recombination

• Integrase

(Int-ase be needed & not need RecA-p )

• Conservative recombination

1. l-encoded integrase (Int): makes staggered cuts in the specific sites

2. Int and IHF (integration host factor encoded by bacteria): recombination and insertion

3. l-encoded excisionase (XIS): excision of the phage DNA

Site-specific recombination:bacteriophage insertion

Site-specific recombination:bacteriophage insertion

attP of λ p O p’

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)

Site-specific recombination: Antibody diversity

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)

250 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）

1. Requires no homology between sequences nor site-specific

2. Relatively inefficient3. Require Transposase encoded by the transpo

son ( 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

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…