Lecture 27

1 / 53 J IANGSU UNIVERSITY

Lecture 27

Chapter 10 Recombination

10.1 Homologous Recombination

10.1.1 Mechanism for Crossing-Over

10.1.2 Mechanism for Double-

Stranded Break Repair

10.1.3 The RecBCD pathway

10.1.4 Gene Conversion

10.2 Non-homologous Recombination

10.2.1 Transposons

10.2.2 Retrotransposons

10.2.3 Bacteriophage λ integration

第 10 章重组10.1 同源重组 10.1.1 交换机理 10.1.2 双链断裂修复机理 10.1.3 RecBCD 途径 10.1.4 基因转换

10.2 非同源重组 10.2.1 转座子 10.2.2 反转录转座子 10.2.3 λ 噬菌体的整合


Review of Lecture 26

A1 → Homologous Recombination 同源重组

A2-1 → Resolution of the Holliday junction to create

a crossover 产生交换的 Holliday 交叉拆分A2-2 → Resolution of Holliday junctions for

non-crossover 不产生交换的 Holliday 交叉拆分

A3 → The RecBCD Pathway / RecBCD 途径


A1 → Homologous Recombination 同源重组

Homologous recombination: Recombination between DNA molecules with similar DNA sequences.

同源重组：在具有相似 DNA 序列的 DNA 分子之间发生的重组。


A2-1 → Resolution of the Holliday junctionto create a crossover

产生交换的 Holliday 交叉拆分


A2-2 → Resolution of Holliday junctionsfor non-crossover

不产生交换的 Holliday 交叉拆分


A3 → The RecBCD Pathway RecBCD 途径

RecBCD pathway: The major homologous recombinationpathway in E. coli, initiated by the RecBCD protein.

RecBCD 途径：大肠杆菌中一种由 RecBCD 蛋白启动的主要的同源重组途径。

RecBCD protein: The protein that nicks one of the DNA strands at a chi site to initiate homologous recombination in E. coli.

RecBCD 蛋白：大肠杆菌中能在 DNA 的 χ位点产生切口的蛋白质。


Questions for Lecture 27

Q1 → What is gene conversion?

什么是基因转换？

Q2 → What are transposons?

什么是转座子？

Q3 → What are retrotransposons?

什么是反转录转座子？


Vocabulary of Lecture 27 (1/1)

site-specific recombinationattachment site

integraseexcisionase

integration host factor, IHFtransposase

resolvasereplicative transposition

conservative transpositionstaggered ends

direct repeatretrotransposon

位点特异性重组附着位点整合酶切除酶整合宿主因子转座酶解离酶复制型转座保守型转座交错末端同向重复（序列）反转录转座子


Q1 → What is gene conversion?什么是基因转换？


A1 → 10.1.4 Gene Conversion / 基因转换

Gene conversion: Consequence of homologous recombination, in which hybridization between different alleles causes one allele to be converted into the other. This occurs by mismatch repair.

基因转换：同源重组的后果，在不同等位基因之间的杂交使其中的一个等位基因被转换成了另一个等位基因。这是由错配修复引起的。

Alleles: Versions of the same gene that differ slightly in function and sequence.

等位基因：同一基因的不同版本，它们之间在功能和序列上稍有不同。


Homologous, not identical同源的，而不是相同的

5’

3’

5’

3’

3’

5’

3’

5’

A double strand cut made by enzyme Spo11Chromosome A

Chromosome B

5’

3’

5’

3’

3’

5’

3’

5’

GTTA

GCTA

Homologous, not identical.

3’5’

5’3’

3’5’

5’3’

Chapter 10, page 225


Gene Conversion / 基因转换

5’3’

3’5’

5’3’

3’5’

5’3’

5’3’

3’5’3’5’


Neurospora crassa 粗糙脉孢菌

+

+m

m

Meiosis I减数分裂 I Meiosis II

减数分裂 II Mitosis有丝分裂

+

+

m

m

+

+

m

m

m

m

m

m

+

+

+

+

4 : 4


Neurospora crassa 粗糙脉孢菌

www.nigms.nih.gov/Initiatives/Models/

www.neurosporagenome.org/

www.neurosporagenome.org/

4 : 4 5 : 3 6 : 2


10.2 Non-homologous Recombination非同源重组

10.2.1 Transposons 转座子10.2.2 Retrotransposons 反转录转座子10.2.3 Bacteriophage λ integration

λ 噬菌体的整合

Non-homologous recombination: Rearrangement of DNA regions that are not similar (homologous) to each other.

非同源重组：互相不相似的（即不同源的） DNA 区域之间的重排。


Q2 → What are transposons?什么是转座子？

MercuryVenus

Earth

Mars

JupitorSaturn

UranusNeptune


A2 → 10.2.1 Transposons / 转座子

GGTACTCTCCCCATGAGAGG

AATCGCGGTTAGCGCC

CCGCGATTGGCGCTAA


Insertion sequence

Direct repeats

Inverted repeats

Genes codingfor transposase

Transposons: DNA elements that can change positions, or locations, in the genome.

转座子：基因组中能改变位置或位点的 DNA 元件。

Insertion sequences: Simple prokaryotic transposons that insert themselves into random sites on a piece of DNA.

插入序列：简单的原核生物转座子。它们是一些能使自身插入到一段 DNA随机位置中的序列。


Complex transposons / 复杂转座子

23 722 23 23 722 23

AntibioticresistanceIS1 IS1

AntibioticresistanceIS1 IS1

Inverted repeat orientation: e.g. Tn10

Direct repeat orientation: e.g. Tn9

23 722 23 23 722 23


More complex transposons / 更复杂的转座子

Organization of the Tn3 transposon转座子 Tn3 的组成

Transposase转座酶

Resolvase解离酶

β-lactamaseβ 内酰胺酶

(Ampr)

IR IR

tnpA tnpR bla

res


Transposition / 转座

What is the other name(s) for transposon?


Simplified mechanism of transposition简化的转座机理

GTACCATGATGCATGGTACTAC

GTACCATGATC-3’

3’-GATGGTACTAC

Make staggered cuts at target site GTACCATGAT

CG

ATGGTACTAC

GTACCATGATCATGGTACTA

TACCATGATGATGGTACTAC

Insert transposon

Repair synthesis and seal gaps

Direct repeats


Direct repeats / 同向重复序列

Direct repeats: Sequences that are exactly the same, and present in the same orientation.

同向重复序列：完全相同并以相同方向出现的序列。

Inverted repeats: Sequences that are the same if you take the complement of one and read it backwards.

反向重复序列：互补序列从反方向读与它自身相同的序列。


Replicative transposition / 复制型转座


Conservative transposition / 保守型转座


Q3 → What are retrotransposons?什么是反转录转座子？


A3 → 10.2.2 Retrotransposon反转录转座子

LTR-Retrotransposons LTR 反转录转座子Non-LTR retrotransposons 非 LTR 反转录转座子

Retrotransposon: A transposable DNA element that transposes via a retrovirus-like mechanism.

反转录转座子：一种以类似于反转录病毒机理进行转座的 DNA 元件。

Retrovirus: An RNA virus whose replication depends on formation of a provirus by reverse transcription.

反转录病毒：一种 RNA 病毒，其复制依赖于通过反转录形成的原病毒。


LTR-Retrotransposons / LTR 反转录转座子

Long terminal repeats (LTRs) : Regions of several hundred base pairs of DNA found at both ends of the provirus of a retrovirus.

长末端重复序列：在反转录病毒的原病毒两端发现的由几百个碱基对组成的DNA 区域。


Non-LTR retrotransposons非 LTR 反转录转座子

p40

Transcription

AA- -A

AA- -A

Export

TranslationRNP assembly

━ AA- -A━EN RT

ORF1 ORF2

AA- -A

AA- -A

AA- -A

5’

5’

5’

5’

p40

ORF2

ORF2

T T- -T

Import or entry

during mitosis

?

━ AA- -A

Integration oftruncated L1at new chro-mosomal siteNucleus

Cytoplasm

Weaver’s Molecular Biology, 3rd Edition, Figure 23.31


10.2.3 Bacteriophage λ integrationλ 噬菌体的整合

Lysis Lysogeny


Phage integration and excision

attP: attachment sites of phageattB: attachment sites of bacterium


Now showing

Gene Conversion

File: biophoto7 \ Introduction to Genetic Analysis \ Genetic Consequences of Meselson-Radding Model


Gene Conversion (1/5)

In this cross of + X m, th

e m site corresponds to

a mutated nucleotide pai

r. If, by chance, m beco

mes part of a heterodupl

ex, the Meselson-Raddin

g model predicts aberra

nt ratios of the + : m alle

les.



In most cases, m will

not be part of a heter

oduplex, so two of th

e four meiotic produc

ts will carry the muta

tion. After one round

of mitosis, the resulti

ng octad （八联体）has a 4:4 ratio of + :

m.



At low frequency, an asymmetric heteroduplex including the m site can be generated by strand invasion, as postulated by the Meselson-Radding model. A crossover event without branch migration generates one +/m heteroduplex.



If the +/m mismatched b

ase pair is not repaired,

a 5:3 octad ratio is prod

uced.



If the m strand of the mi

smatched DNA is repair

ed to +, a 6:2 octad ratio

results.


Now showing again


Now showing

Replicative transposition

File: biophoto7 \ Introduction to Genetic Analysis \ Replicative transposition. (5’)


Replicative transposition (1/9)

Transposition is the movement of genetic elements, called transposons, between plasmids or genomes.



Shown here is a mechanism of replicative trans-position by the E. coli Tn3 transposon. A copy of Tn3 will be generated in the target plasmid.



The transposon is flanked by inverted repeat sequences and consists of several genes. One of these genes encodes the transposase enzyme.



In the target plasmid, transposase acts at the region of insertion.



The target plasmid is cleaved by transposase to yield staggered ends nine nucleotides apart.



Transposase also cleaves the plasmid containing the transposon at the ends of the transposable element. These ends are joined with the ends of the target plasmid.



The single-stranded regions of DNA are replicated.



The resulting plasmid is called a cointegrate（共整合体） . It contains the original transposon and a new copy.



The cointegrate is resolved into the original plasmid and the target plasmid by an enzyme called resolvase. The transposon contains a sequence called the internal resolution site, or IRS, which is necessary for resolution.


Now showing again


Essentials of Lecture 27 / 第 27 讲要点

A1 → Gene Conversion / 基因转换

A2 → Transposons / 转座子

A3 → Retrotransposon / 反转录转座子


A1 → 10.1.4 Gene Conversion / 基因转换

Gene conversion: Consequence of homologous recombination, in which hybridization between different alleles causes one allele to be converted into the other. This occurs by mismatch repair.

基因转换：同源重组的后果，在不同等位基因之间的杂交使其中的一个等位基因被转换成了另一个等位基因。这是由错配修复引起的。

Alleles: Versions of the same gene that differ slightly in function and sequence.

等位基因：同一基因的不同版本，它们之间在功能和序列上稍有不同。


A2 → 10.2.1 Transposons / 转座子


AATCGCGGTTAGCGCC

CCGCGATTGGCGCTAA


Insertion sequence

Direct repeats

Inverted repeats

Genes codingfor transposase

Transposons: DNA elements that can change positions, or locations, in the genome.

转座子：基因组中能改变位置或位点的 DNA 元件。

Insertion sequences: Simple prokaryotic transposons that insert themselves into random sites on a piece of DNA.

插入序列：简单的原核生物转座子。它们是一些能使自身插入到一段 DNA随机位置中的序列。


A3 → 10.2.2 Retrotransposon反转录转座子

LTR-Retrotransposons LTR 反转录转座子Non-LTR retrotransposons 非 LTR 反转录转座子

Retrotransposon: A transposable DNA element that transposes via a retrovirus-like mechanism.

反转录转座子：一种以类似于反转录病毒机理进行转座的 DNA 元件。

Retrovirus: An RNA virus whose replication depends on formation of a provirus by reverse transcription.

反转录病毒：一种 RNA 病毒，其复制依赖于通过反转录形成的原病毒。


Lecture 28

Appendix D

Experimental Techniques

D.1 DNA manipulation

techniques

D.2 Manipulating Proteins

D.3 The Function of Genes

and their Proteins

附录 D

实验技术

D.1 DNA 操作技术

D.2 操作蛋白质

D.3 基因及其蛋白质的功能

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Lecture 27

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