第 31 章 dna 的重组
DESCRIPTION
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. Recombination. Homologous recombination Site-specific recombination Transposition. Mutation Relevance - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/1.jpg)
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
![Page 2: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/2.jpg)
Homologous recombination Site-specific recombination Transposition
RecombinationRecombination
Mutation RelevanceAn important reason for variable DNA sequences among different populations of the same species
![Page 3: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/3.jpg)
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
![Page 4: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/4.jpg)
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
![Page 5: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/5.jpg)
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)
![Page 6: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/6.jpg)
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
![Page 7: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/7.jpg)
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:
![Page 8: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/8.jpg)
• 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
![Page 9: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/9.jpg)
Diploid eukaryotes: crossing over1. Homologous chromosomes line up in meiosis (when)
2. The nonsister chromatids exchange equivalent sections (what)
![Page 10: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/10.jpg)
Holliday Intermediate structure
![Page 11: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/11.jpg)
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)
![Page 12: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/12.jpg)
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’
![Page 13: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/13.jpg)
3. RecA-ssDNA filaments search the opposite DNA duplex for corresponding sequence (invasion).
4. form a four-branched Holliday structure
5. Branch migration
![Page 14: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/14.jpg)
6. Resolving Holliday junction
![Page 15: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/15.jpg)
![Page 16: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/16.jpg)
RuvAB is an asymmetric complex that promotes branch migration of a Holliday junction.
![Page 17: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/17.jpg)
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
![Page 18: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/18.jpg)
![Page 19: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/19.jpg)
Rec.A-p
Hydrogen bond reform
heterodulpex
![Page 20: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/20.jpg)
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
![Page 21: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/21.jpg)
![Page 22: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/22.jpg)
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
![Page 23: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/23.jpg)
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)
![Page 24: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/24.jpg)
Site-Dependent
Specific Recombination
• Integrase
(Int-ase be needed & not need RecA-p )
• Conservative recombination
![Page 25: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/25.jpg)
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
![Page 26: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/26.jpg)
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)
![Page 27: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/27.jpg)
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
![Page 28: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/28.jpg)
![Page 29: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/29.jpg)
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
![Page 30: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/30.jpg)
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)
![Page 31: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/31.jpg)
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)
![Page 32: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/32.jpg)
Simplified Transposition process
Simplified Transposition process
![Page 33: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/33.jpg)
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…
![Page 34: 第 31 章 DNA 的重组](https://reader034.vdocuments.mx/reader034/viewer/2022050617/56814f21550346895dbcb1c2/html5/thumbnails/34.jpg)