Recombination of Dna

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DNA Recombination

Roles

Types Homologous recombination in E.coli

Transposable elements



Biological Roles for Recombination

1. Generating new gene/allele combinations

(crossing over during meiosis)

2. Generating new genes (e.g., Immuno-

globulin rearrangement)

3. Integration of a specific DNA element

4. DNA repair



Practical Uses of Recombination

1. Used to map genes on chromosomes

(recombination frequency proportional

to distance between genes)

2. Making transgenic cells and organisms



Map of Chromosome I of

Chlamydomonas reinhardtii

Chlamydomonas Genetics Center

cM = centiMorgan; unit of recombination frequency

1 cM = 1% recombination frequency



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 fromone site to another, usually little sequence

similarity involved



Examples of Recombination

Fig. 22.1



Holliday Model

R. Holliday (1964)

- Holliday Junctions

form during

recombination

- HJs can be resolved2 ways, only one

produces true

recombinant

molecules

Fig. 22.2 patch



Fig. 22.5 a-e

The recBCD

Pathway of

Homologous

Recombination

Part I: Nicking and

Exchanging



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 byrecBCD 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 singlestrand 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

Fig. 22.5 f-h



recBCD Pathway of Homologous Rec.

Part II: Branch Migration and Resolution

1. Nicks are sealed Holliday Junction2. Branch migration (ruvA + ruvB)

3. Resolution of Holliday Junction (ruv C )



R ecBCD : A complex enzyme

R ecBCD has:

1. Endonuclease subunits (recBCD) that cut

one DNA strand close to Chi sequence.

2. DNA helicase activity (recBC subunit) and

3. DNA-dependent ATPase activity

± unwinds DNA to generate SS regions

Activity 2 and 3 linked



R ecA

38 kDa protein that polymerizes onto SS DNA 5¶-3¶

Catalyzes strand exchange, also an ATPase

Also binds DS DNA, but not as strongly as SS

Fig. 22.6



R ecA Function Dissected

3 steps of strand exchange:

1. Pre-synapsis: recA coats single strandedDNA (accelerated by SSB, get more relaxed

structure, Fig. 22.8)2. Synapsis: alignment of complementary

sequences in SS and DS DNA (paranemicor side-by-side structure)

3. Post-synapsis or strand-exchange: SS DNAreplaces the same strand in the duplex toform a new DS DNA (requires ATPhydrolysis)



R uvA and R uvB

DNA helicase that catalyzes branch migration

R uvA tetramer binds to HJ (each DNA

helix between subunits)

R uvB 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



http://www.sdsc.edu/journals/mbb/ruva.html



R uv C : 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 reachconsensus sequence!



R uv C

bound to

Holliday junction

Fig. 22.31a



Transposable Elements

(Transposons)

DNA elements capable of moving ("transposing")

around the genome

Discovered by Barbara McClintock, largely from

cytogenetic studies in maize, but since foundin most organisms

She was studying "variegation" or sectoring in

leaves and seeds

She liked to call them "controlling elements³

because they effected gene expression in

myriad ways



Mutant Kernel Phenotypes

Pigmentation mutants ± affect anthocyanin pathway

± elements jump in/out of transcriptionfactor genes (C or R)

± sectoring phenotype - somatic mutations ± whole kernel effected - germ line

mutation

Starch synthesis mutants- stain starch with iodine, see sectoring in

endosperm



Start with mutant kernels defective in starch synthesis (endosperm

phenotypes) or anthocyanin synthesis (aleurone and pericarp

phenotypes).

Some maize phenotypes caused by transposable

elements excising in somatic tissues.



Somatic Excision of Ds from C

Fig. 23.19

SectoringWild type Mutant



Other Characteristics of McClintock's

Elements

Unstable mutations that revert frequently but often

partially, giving new phenotypes.

Some elements (e.g., Ds) correlated with

chromosome breaks.

Elements often move during meiosis and

mitosis. Element movement accelerated by genome

damage.



Molecular analysis of transposons

Transposons isolated by first cloning a gene thatthey invaded. A number have been cloned this way,

via "Transposon trapping³.

Some common molecular features: ± Exist as multiple copies in the genome

± Insertion site of element does not have extensive

homology to the transposon

± Termini are an inverted repeat ± Encode ³transposases´ that promote movement

± A short, direct repeat of genomic DNA often

flanks the transposon : ³Footprint´



Ac and Ds

Ds is derived from Ac by internal deletions

Ds is not autonomous, requires Ac to move

Element termini are an imperfect IR Ac encodes a protein that promotes

movement - Transposase

Transposase excises element at IR, and also

cuts the target



Structure of Ac and Ds deletion

derivatives

Fig. 23.20D

s is not autonomous, requires Ac to move!



How duplications

in the target site

probably occur.

Duplication

remains whenelement excises,

thus the

Footprint.



utator (A Retrotransposon)

Discovered in maize; differs significantly from Ac by structure and transposing mechanism

Autonomous and non-autonomous versions;

many copies per cell contains a long terminal IR (~200 bp)

transposes via a replicative mechanism,instead of a gain/loss mechanism

A ³retrotransposon´ ± Similarities with retroviruses

± move via an RNA intermediate

± encode a reverse transcriptase activity



Fig. 7.34 in Buchanan et al.



Control of Transposons Autoregulation: Some transposases are

transcriptional repressors of their own

promoter(s) e.g., TpnA of the S pm element

Transcriptional silencing: mechanism not well

understood but important; correlates with

methylation of the promoter



Biological Significance of

Transposons

They provide a means for genomic change

and variation, particularly in response to

stress (McClintock¶s "stress" hypothesis)(1983 Nobel lecture, Science 226:792)

or just "selfish DNA"?

No known examples of an element playing a

normal role in development.

Recombination of Dna

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