Chapter 11 DNA recombination

and Transposon

Examples of Recombination

交互的

DNA Recombination

Roles Types Homologous recombination in E.coli Transposable elements

Biological Roles for Recombination

1. Deleterious mutations would accumulate in each chromosome. Recombination generates genetic diversity 多样性

2. Generating new gene/allele combinations (crossing over during meiosis)

3. Integration of a specific DNA element

4. Role in DNA damage and repair

5. Gene regulation

Practical Uses of Recombination

1. Used to map genes on chromosomes (recombination frequency proportional to

distance between genes)

2. Making transgenic cells and animals

Types of Recombination

1. Homologous - occurs between sequences that are nearly identical (e.g., during meiosis)

2. Site-Specific - occurs between sequences with a limited stretch of similarity; involves specific sites

3. Transposition – DNA element moves from one site to another, usually little sequence similarity involved

Homologous Recombination: Homologous Recombination: The Holliday model (1964) The Holliday model (1964)

Two homologous duplexes are alignedTwo homologous duplexes are aligned

Strand exchange leads to an Strand exchange leads to an intermediate with crossed strandsintermediate with crossed strands

This branch can move: Branch This branch can move: Branch migration (~30bp/s)migration (~30bp/s)

The branch is resolved The branch is resolved （拆分） （拆分） by by cleavage and sealingcleavage and sealing

Holliday Model

R. Holliday (1964)

- Holliday Junctions form during recombination

- HJs can be resolved 2 ways, only one produces true recombinant molecules

Patch recombination 片段重组体

Splice recombination 拼接重组体

Holliday 中间体

Isomerization (Holliday 的异构化 )

Resolution

Holliday 结构一经生成即可不断地处于异构化－－异源双链 hetero duplex DNA

（ 1 ）

（ 2 ）

（ 1 ）（ 2 ）

◘ Holliday resolution

产生含异源双链的片段重组体

产生拼接重组体

3‘5‘

5‘3‘

Meselson-Radding 模型单链入侵模型（链转移模型）

置换

侵入

Loop 切除

同化 异构化 分支迁移

5’

切割

Double Double strand strand break break modelmodel

双链断裂修复模型

Double Double strand strand break break modelmodel

Homologous recombination in E.coli

◘ This process involved the exchange of

homologous regions between two molecules

Conjugation 接合Transformation 转化Transduction 转导Cell fusion 细胞融合

Chi site and RecBCD ptotein

E.coli also perform recombination,

the nick site in two homologous

DNA duplexes created by RecBCD

are near a specific sequence

called Chi site.

Chi site

◘ A Chi site is a short stretch of DNA near which homologous recombination is unusually likely to occur.

◘ In E. coli, the sequence is 5'-GCTGGTGG-3'. 1/5~10kb

◘ Chi serves as a signal to the RecBCD helicase-exonuclease.

RecBCD : A complex enzyme

◘ RecBCD enzyme is both a helicase and a nuclease

◘ RecBCD recognizes a specific sequence of Chi site.

◘ RecBCD cut one DNA strand close to Chi sequence

凡能从多核苷酸链的末端开始水解核酸的酶称为核酸外切酶凡能从多核苷酸链中间开始水解核酸的酶称为核酸内切酶而能识别特定的核苷酸顺序 , 并从特定位点水解核酸的内切酶称为限制性核酸内切酶

•RecBCD binds an end of linear dsDNA

•RecD helicase travels on the strand with a 5' end and RecB on the strand with a 3' end

•RecB is slower than RecD, so that a ssDNA loop accumulates ahead of RecB

•This produces DNA structures with two ss tails and one ss loop

•ss tails can anneal to produce a second ss loop complementary to the first one; such twin-loop structures were referred to as “rabbit ears.” •When RecBCD encounters a Chi site on the 3' ended strand, unwinding pauses and digestion of the 3' tail is reduced.

• The RecA protein is then actively loaded onto the 3' tail by RecBCD and form RecA-ssDNA filaments.

• RecA catalyzes branch migration and makes it possible to complete recombination

recBCD Pathway of Homologous Recombination

The recBCD Pathway of Homologous Recombination

Part I: Nicking and Exchanging

Assimilation

同化

recBCD Pathway of Homologous Recombination Part I: Nicking and Exchanging

1. A nick is created in one strand by recBCD at a Chi sequence (GCTGGTGG), found every 5000 bp.

2. Unwinding of DNA containing Chi sequence by recBCD allows binding of SSB and recA.

3. recA promotes strand invasion into homologous DNA, displacing one strand.

4. The displaced strand base-pairs with the single strand left behind on the other chromosome.

5. The displaced and now paired strand is nicked (by recBCD?) to complete strand exchange.

recBCD Pathway of Homologous Recombination

Part II: Branch Migration and Resolution

Generation of a Generation of a chi intermediatechi intermediate

Electron Electron micrograph of the micrograph of the

chi formchi form

Without functional RecA protein in E.coli, the exogenous plasmid DNA is left unaltered by the bacteria. Purification of this plasmid from bacterial cultures can then allow high-fidelity PCR amplification of the original plasmid sequence.

RecA38 kDa protein that polymerizes 聚合 onto SS DNA 5’-

3’Catalyzes strand exchange, also an ATPaseAlso binds DS DNA, but not as strongly as SS

RecA Function Dissected

Three steps of strand exchange:1. Pre-synapsis: recA coats single stranded DNA

(accelerated by SSB, get more relaxed structure)

2. Synapsis 联会 : alignment of complementary sequences in SS and DS DNA (paranemic or side-by-side structure)

3. Post-synapsis or strand-exchange: SS DNA replaces the same strand in the duplex to form a new DS DNA (requires ATP hydrolysis)

RecA promotes the assimilation of invading single strands ( 单链同化） into duplex DNA so long as one of the reacting strands has a free

end.

Ruv protein and resolution resolution

of Holliday structuresof Holliday structures

Need three genes in E.coli, ruvA,ruvB and ruvC

a 、 RuvA recognize the junction of Holliday

b 、 RuvB provide energy for migration (ATPase 1

0 ～ 20bp/s ） c 、 RuvC nuclease cut Holliday junction specifically.

RuvA and RuvB

DNA helicase that catalyzes branch migrationRuvA tetramer binds to HJ (each DNA helix

between subunits)RuvB is a hexamer ring, has helicase & ATPase

activity 2 copies of ruvB bind at the HJ (to ruvA and 2 of

the DNA helices)Branch migration is in the direction of recA

mediated strand-exchange

RuvC : resolvase

Endonuclease that cuts 2 strands of HJBinds to HJ as a dimer Consensus sequence: (A/T)TT (G/C)

- occurs frequently in E. coli genome

- branch migration needed to reach consensus sequence!

RuvC bound to Holliday junction

Action of Action of E. coliE. coli proteins in branch proteins in branch migration and resolution of Holliday migration and resolution of Holliday

structuresstructures

Site specific recombinationSite specific recombination

Viruses and transposable elements often integrate Viruses and transposable elements often integrate their genomes into the host chromosometheir genomes into the host chromosome

Site specific recombination is used by both Site specific recombination is used by both eukaryotes and prokaryotes to regulate gene eukaryotes and prokaryotes to regulate gene expression and to increase the organisms genetic expression and to increase the organisms genetic repertoirerepertoire

位点特异性重组

Site specific recombinationSite specific recombination

1 、 Occurs between sequences with a limited stretch of similarity; involves specific sites and proteins 依赖于小范围同源序列的联会，重组也依赖于小范围同源序列的联会，重组也只发生在同源的短序列的范围之内，需要位点特异性的蛋只发生在同源的短序列的范围之内，需要位点特异性的蛋白质分子参与催化白质分子参与催化

2 、 Based on position and direction

λphage integrate and cleavage

1 、 Via Site-specific recombination

2 、 attachment site (att)

◘ E.coli attB BOB’ 23bp

◘ λphage attP POP’ 240bp

◘ “ O” complete

same （ 15bp ）

3 、 Integration

◘ attachment site

attL （ BOP’ ） attR （ POB’ ）◘ Integration site---attB 、 attP

Excision site---attL 、 attR

◘ need integrase Int （ λ encoded ） and integration host factor

溶源性细菌(lysogen)

溶菌周期（ lysis ）

原噬菌体

4 、 Mechanism

◘ Core sequence is “O” 15bp ， A-T rich

◘ exchange within O region ： 7bp

◘ Int Binding site ： attP 240bp 、 attB 23bp

◘ intasome （整合体） : Both Int

and IHF bind to attP and form an

intasome

◘ intasome recognize attB

a 、 attB and attP are identified

by Int

b 、 Homologous sequences is

important for recombination

◘ Int cleavages DNA lead to crosswise pairing holliday J

◘ Recombinant junction are resolved, sealed to generate

intergrated prophage DNA

5 、 The control of integration - excision

Control of the integration-excision reaction depends on:

(1) the forward (insertion) reaction, which requires only Int

among phage-specified proteins

(2) the reverse (excision) reaction, which requires the phage-

coded Int and Xis proteins

Knockout mouse

A genetically engineered mouse in which one or more genes have been turned off through a targeted mutation.

The first knockout mouse was created by Mario R. Capecchi, Martin Evans and Oliver Smithies in 1989, for which they were awarded the Nobel Prize for Medicine in 2007

Generate Knockout mice based on HR

Conditional knockout mice