Transcript recBCD

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:
The Holliday model (1964)
Two homologous duplexes are aligned
Strand exchange leads to an
intermediate with crossed strands
This branch can move: Branch
migration (~30bp/s)
The branch is resolved （拆分） by
cleavage 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
Holliday中间体
Splice recombination
拼接重组体
Holliday结构一经生成即可
不断地处于异构化－－异源双链
hetero duplex DNA
Isomerization (Holliday的异构化)
Resolution
◘ Holliday resolution
（1）
产生含异源双链
的片段重组体
（2）
（2）
（1）
产生拼接重组体
3‘
5‘
切割
5‘
3‘
Meselson-Radding模型
单链入侵模型（链转移模型）
5’
置换
侵入
Loop切除
同化 异构化 分支迁移
双链断裂修复模型
Double
strand
break
model
Double
strand
break
model
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 helicaseexonuclease.
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 Pathway of Homologous Recombination
•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 RecAssDNA filaments.
• RecA catalyzes branch migration and makes it possible to complete
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.
2.
3.
4.
5.
A nick is created in one strand by recBCD at a Chi
sequence (GCTGGTGG), found every 5000 bp.
Unwinding of DNA containing Chi sequence by recBCD
allows binding of SSB and recA.
recA promotes strand invasion into homologous DNA,
displacing one strand.
The displaced strand base-pairs with the single strand left
behind on the other chromosome.
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
chi intermediate
Electron
micrograph of the
chi 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.
RecA
 38 kDa protein that polymerizes 聚合 onto SS DNA 5’-3’
 Catalyzes strand exchange, also an ATPase
 Also 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
of 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
10～20bp/s）
c、 RuvC nuclease cut Holliday junction specifically.
RuvA and RuvB
DNA helicase that catalyzes branch migration
RuvA 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 HJ
Binds 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 E. coli proteins in branch
migration and resolution of Holliday
structures
Site specific recombination
位点特异性重组
Viruses and transposable elements often integrate
their genomes into the host chromosome
Site specific recombination is used by both
eukaryotes and prokaryotes to regulate gene
expression and to increase the organisms genetic
repertoire
Site 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
2、Based on position and direction
λphage integrate and cleavage
1、Via Site-specific recombination
2、attachment site
◘
E.coli
(att)
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
溶菌周期
（lysis）
溶源性细菌
(lysogen)
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 phagecoded 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