Re Combination 2010-SPIN Version

8/8/2019 Re Combination 2010-SPIN Version

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STBP1013

FUNDAMENTALS OF MOLECULAR

BIOLOGY

DNA RECOMBINATION


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WHAT IS RECOMBINATION??

process or set of processes by which

DNA molecules interact with one another

to bring about a rearrangement of the

genetic information or content in an

organism.


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In eukaryotic systems -recombination as the process

that is responsible for crossing-

over during meiosis.

Crossing-over has been well-documented genetically and is

used to map the relativelocations of genes on a

chromosome


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Examples of recombination in prokaryotic

systems are(i) integration of the bacteriophage

lambda prophage,

(ii) recombination of bacterial DNAfollowing conjugation between bacteria

(iii) formation of plasmid multimers


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5

Replication of Bacteriophage- Evidence for

Recombination Events


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Bacteriophage Plaque assay


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Recombination in a bacterial

system was first demonstrated independently by Al

Hershey and Max Delbrck in 1947.

They studied the infection ofE. coli withbacteriophage.

If an E. coli cell was infected at the same time

with two genetically different bacteriophage,

the resulting phage population included

recombinant phage types as well as the

original parental phage types.


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The hlocus determines whether the phage

can grow on a particular strain ofE. coli:

phage that are h-

can infect the strain; phage that are h+ cannot.


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The rlocus is a gene that determines whether

the phage will lyse the host cells rapidly orslowly:

phage that are r - will lyse the host cellsrapidly;

phage that are r + will lyse the host cell slowly.


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In addition, two strains ofE. coli were used in

the experiment:

strain 1 supports growth ofh-

phage but noth+ phage;

strain 2 supports the growth of both phages.


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In the experiment, bacteriophages are plated

on a lawn of bacteria that consists of amixture of both strains ofE. coli.

If a phage can infect both strains of bacteria(i.e. if it is h-) then the resulting plaque will be

clear.

If the phage can infect only one of the two

strains of bacteria (i.e. if it is h+) then the

resulting plaque will be turbid because thenon-infected bacteria will be growing.


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When the experiment is performed, four types

of plaque were observed:

phenotype inferred genotype

Clear and small h-r+

Cloudy and large h+

r

-

Cloudy and small h+r+

Clear and large h-r-

Note:

r

phage will lyse the host cells rapidly;r+phage will lyse the host cell slowlyphage that are h-can infect both E. coli strains;phage that are h+can infect only one E. colistrain.


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Most of the plaques correspond to theparental phenotypes but a significant number

have the recombinant phenotypes.


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Most of the plaques correspond to the

parental phenotypes but a significant number

have the recombinant phenotypes.


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However, when the progeny phage were usedto reinfect E. coli so as to examine their

phenotype, a low but definite percentage of

the resulting plaques were found to contain

two different types of phage although onlyone type had been expected.

This implies that some of the progeny phage

were not genetically homogeneous.


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This observation can be explained by models

of recombination that allow for heteroduplexforms to be generated.

Al Hershey and Max Delbrck shared the 1969

Nobel prize in Medicine & Physiology with

Salvador Luria for their discoveries concerning

"the replication mechanism and the genetic

structure of viruses"


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The Meselson - Weigle Experiment

In the simplest sense, recombination is an

exchange of both strands between two DNA

molecules:


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Note: each line in the above cartoon figurerepresents one strand of a DNA double helix.


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This representation implies that both strands

of each molecule must be broken and then

rejoined. This was first demonstrated by an

experiment performed by Matt Meselson and

Jean Weigle in 1961.


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Meselson and Weigle infected E. coli cells at

the same time with phage from two different

stocks of bacteriophage lambda.

One stock had been prepared by growing the

bacteriophage lambda c-mi- in cells grown in

medium containing heavy isotopes of carbon

(13C) and nitrogen (15N).


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Note: each line in the above figure represents a phage

chromosome, i.e. a double helical DNA molecule.


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After infection, the progeny phage wereisolated and banded on a CsCl gradient.

A broad band of phage particles were

found on the gradient.

Non-recombinant phage were found, asexpected, at two well-defined densitiescorresponding to the parental light andheavy phages.

Recombinant phage were found -surprisingly - at all intermediate densities

between these two.


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They also followed the course of the infection

using two genetic markers, c and mi, which

were located near one end of the lambda

chromosome.


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Note: each line in the above figure represents aphage chromosome, i.e. a double helical DNA

molecule.


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When the phenotypes of the intermediate

density phage particles were analyzed,

recombinant phage that were c-mi+ were

found near the band of "heavy" phage while

recombinant phage that were c+mi- were

found near the band of "light" non-

recombinant phage.


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3 general types of recombination

1. Homologous genetic recombination

2. Site-specific recombination

3. Illegitimate recombination


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1. Homologous Recombination

Also known as general recombination or generalhomologous recombination

The exchange of genetic material between two

molecules that share a large degree of identity with oneanother.

This is the type of recombination that is required duringmeiotic crossing over, for bacteriophage recombination,

for recombination following bacterial conjugation, andduring the formation of plasmid multimers


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Homologous Recombination

Exchange of DNA sequences betweenDNA molecules that contain identical or

nearly identical sequences along their

length.

The region to be recombined is knownas homology between sequences can be

as few as 50-100 bp or as much as the

whole chromosome


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The requirement for homologous recombination

1. Two DNA sequences with similar or almost identicalbase pair sequence (homologous sequence)

2. The ability to form stable hydrogen bonds between the

bases on one strand of DNA sequence and the baseson the complementary strand on the other DNA

sequence

3. Proteins needed to carry out recombination. These

proteins include those make two DNA sequence to

stay close to each other, enzymes that break

phosphodiester bonds (endonuclease or exonuclease)

and enzymes that rejoin phosphodiester bond (ligase).


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Early Models for Homologous

Recombination


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RecA binds single-stranded DNA,promote base pairing

RecA binds to single stranded DNA

in the presence of ATP to form

RecA-ssDNA.

A RecA-ssDNA filament can bind

and unwind (by breaking hydrogenbonds) another double stranded

DNA promoting base pairing of the

nucleotides in the ssDNA with

nucleotides in the complementary

strand of the homologous dsDNA

Important protein in E. coli for homologous

recombination

RecA

RecA


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RecB, RecC and RecD also known

as RecBCD or Exonuclease V. Has

various function Nuclease - nick dsDNA, ssDNA.

Created the ssDNA , first

function needed in homologous

recombination

Helicase activity separate the

strands of dsDNA.

ATPase activity - hydrolyses ATP

Other protein RecE, RecF, RecG,

RecJ, RecN, RecO, RecQ, RecR, RecT,

RusA, RuvA, RuvB, RuvC various

functions such as exonuclease,

endonuclease, ATPase, dsDNA

helicase, binds to Holliday junction,

etc


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RecBCD enzyme binds to a blunt-endDNA from a double-stranded DNA break.

It unwinds the dsDNA and preferentiallydegrades the 3-terminating strand (top

strand).

Interaction with (chi site) results inattenuation of the 3 5 nuclease

activity, activate the weaker 5 3

nuclease activity, and the facilitated

loading of RecA protein onto the -

containing ssDNA.

Proposed function of RecBCD and RecA in homologous recombination in E. coli


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Model for Homologous Recombination


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The intermediate that is formed is called a

Holliday intermediate or Holliday structure.

The shape of this intermediate in vivo is

similar to that of the greek letter chi, hence

this is also called a chi form.


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Resolve the structure.

There are two ways in which this can happen:

If the same strands are cleaved a second time

then the original two DNA molecules are

generated:


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If the other strands are cleaved, thenrecombinant molecules are generated:


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Holliday Structure

Name after Robin Hollidaywho in 1964, proposed a

model for homologous

recombination and re-established by David

Dressler and HuntingtonPotter in 1976


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Potter & Dressler's evidence for

the Holliday Model In 1976, David Dressler and Hunt Potter

published the results of a series of

experiments that demonstrated the validity of

the Holliday model of recombination.


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They used E. coli cells containing the colicin E1

derived plasmid, pMB9.

This plasmid was one of the very earliestplasmids developed for cloning in Herbert

Boyer's laboratory.


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Normally, E. coli contain about 20 copies of

this plasmid per cell.

However, if the cells are exposed tochloramphenicol then, although chromosomal

replication stops, plasmid replication does not

and the number of plasmid molecules

increases to 1000 copies per cell.


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With so many more copies of the plasmid in

the cell, the chances of recombination

increase as does the probability of observing a

recombination intermediate.


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When plasmid was isolated from the cells,

purified by CsCl gradient centrifugation, and

observed in the electron microscope, a

number of candidates for intermediates were

observed.


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These all had the appearance of "figure 8"

structures. However, there are 3 possible ways

such structures might arise:

I. as a double-sized circular plasmid twisted

over on itself.

II. as two interlocking circular plasmid

molecules.

III. as a genuine recombination intermediate.


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In order to distinguish between the three

possibilities, Potter and Dressler digested their

plasmid preparations with EcoRI.

This enzyme will generate monomer sized

linear molecules from either of the first two

possible structures.

However, it will generate unique chi-shapedstructures from the third.


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When they did this, Potter and Dressler foundthat between 0.5% and 3% of the moleculesthey observed were chi-shaped structures.


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The molecules were symmetrical in that the

opposite arms were identical lengths and had

identical denaturation patterns.

Finally, they saw no such structures if they

prepared their plasmids from recA- strains of

E. coli.


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From this evidence they concluded:

. . . the intermediates we have observed in

the electron microscope provide physical

evidence in support of the recombination

intermediate postulated by Holliday on

genetic grounds.


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Site-specific Recombination

Involves a protein called

recombinase, that acts on both

participating DNA sequences at a very

specific sequence on the nucleotides.

The sequence is often short and mustbe present on both DNA segments

Different site-specific recombinasewill recognise different sequence.

Site specific recombination can takeplace between two separate DNAmolecules as long as they have the

same specific site (intermolecular).


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It can also take place withinthe same DNA molecule

(intramolecular) the specific

site must present at least

twice in the molecule

Described as conservatives

due: No nucleotide are lost

DNA replication is not

required. Involves in

breakage and rejoining of

DNA strands . ATP is not required for

site specific recombination


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Site-specific Recombination: Integration of Bacteriophage chromosome into E. coli

The molecular event for

integration and excision

of bacteriophage .

To integrate the

genomeinto the E.coli

chromosome, two specific

sites (attP and attB) and

the activities of Int and

E. coli IHF are required


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Illegitimate recombination

There are a number of other geneticexchanges which do not fall into any of the

above classes - hence their name: illegitimate

recombination. Illegitimate recombination is a broad term

designating recombination events that are

independent of RecA (Michel 1999).

Include spontaneous DNA rearrangement such

as deletions, duplications and formation of

specialized phage.


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Scenarios of illegitimate recombination between close direct repeats and SSRs. Black boxesrepresent start and stop codons of the original gene, grey boxes represent strict repeats, and

light grey boxes represent regions of weaker homology. Dashed lines indicate that deletionsand duplication may induce frameshifts and therefore produce ORFs of very different length.(A and B) Duplication/deletion of the repeat and the region between occurrences. (C) Theregions of non-strict similarity become similar after conversion. (D and E) Increase/decrease inthe number of motifs of the SSR. Homologous recombination between long repeats closelyfollows the scenarios of (A), (B) and (C), except that large duplications are unstable and largedeletions are strongly counter selected. Thus, conversions or reciprocal translocations are themost frequent outcome of homologous recombination between long distant repeats.


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The process termed as "homology-facilitated

illegitimate recombination" (HFIR) to indicate

the essential role of homologous anchor

sequences.

For example, HFIR has been shown tomediate the transfer of genes from transgenic

tobacco plastids (trnL und ycf5) to

Acinetobacter sp. equipped with a truncated

aadA gene.

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Re Combination 2010-SPIN Version