1. General homologous recombination

2. Site-specific recombination

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2

2 types in bacteria General recombination

• Require long (>50 bp) sequence homology• RecA-dependent

Site-specific recombination• Require very short (<5 bp) sequence homology;• Special site recognition• RecA-independent but require specialized proteins

E.g. transposition

Genetic exchange takes place between 2 pieces of homologous DNA sequences

May be intra- or inter-molecular events

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Heteroduplexformation at the site of crossover

No alteration of nucleotide sequences at the site of exchange

New recombinant DNA molecules are produced

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Recombination is initiated by a nick in one strandRecA, RecBCD RecA Ligase

Single stranded DNA, coated by RecA, invades homologous duplex

Holliday junction

Resolution & Ligation

Single-strand invasion model (Messelson-Radding model)

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1. Limited degradation at double-strand break by a 5’3’ exonuclease to create protruding single-stranded 3’ tails

2. Single-stranded DNA are recognized by RecA protein which initiates homology search in the other chromosome

3. ATP-dependent strand exchange occurs followed by DNA synthesis and ligation

4. Branch migration of Holliday junctions

5. Resolution by strand cutting

Double strand break model

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Involvement of homologous

recombination in repair of ds breaks

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binds to single-stranded DNA (produced from nicks, dsbreaks or gaps)

mediates ATP-dependent strand invasion and exchange catalyzes branch migration

352 a.a.

6 RecA monomers per turn

Spooling in

Spooling out

RecA is a long filamentous multisubunit protein complex that coats DNA in vivo

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Rad51 protein in yeast, mice, humans In humans, Rad51 function together with

accessory proteins, e.g. BRCA1 and BRCA2, which are mutated in human breast cancers

Human Rad51 was shown to be involved in the resolution of Holliday junctions [Science (2004) 303:243-246]

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helicase

3’5’ & 5’3’ exonucleases

ss DNA is coated by RecA for homologous recombination

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~1000 chi sites (5'-GCTGGTGG-3’) are present on the E. coli chromosome

Chi sites are “hotspots” for general recombination

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RuvA22 kD protein which binds to

RuvB and Holliday junctions

RuvB37 kD DNA-dep ATPase

which drives branch migration

RuvC 19 kD nuclease which

resolves Holliday structures (resolvase)

DNA ligase

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RuvC cleaves DNA strands at

the H-J

Ligase seals the nicked strands

RuvA and RuvBrecognize H-J and promotes branch

migration

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RecA mediated single-strand invasion

Heteroduplex DNA is formed

Gene conversion is non-reciprocal exchange

Only small sections of DNA or part of a gene undergoes gene conversion

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Mismatched DNA in a heteroduplex are recognized and removed by the DNA repair enzymes and replaced with a copy of the complementary strand

NO GENE CONVERSION

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Homologous recombination pathways are shared by DNA repair mechanisms

Occurs predominantly in mitotic cells

Occurs predominantly in meiotic cells

Homologous strand exchange initiated by double strand break

5’5’

5’3’

5’3’

3’

3’

5’3’

5’5’

5’

3’3’

3’

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General or homologous recombination in bacteria is RecA-dependent and requires large regions of homology

Other enzymes involved include RecBCD, RuvA, RuvB, RuvC, DNA ligase

Mechanisms of general recombination can be explained using

• Single-strand invasion model (initiated by a nick)• Double-strand break model (initiated by a ds break)

Branch migration determines the extent of heteroduplex formation

Isomerization and resolution of Holliday structures determine whether “splices” or “patches” are obtained

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General recombination together with DNA repair can produce gene conversion

Eukaryotes also have RecA homologs which participate in general recombination

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1. Transpositional site-specific recombination

2. Conservative site-specific recombination

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Class description and structure

Genes in complete element

Mode of movement Examples

DNA-only transposons

Short inverted repeats at each end

Encodes transposase

Moves as DNA, either excising or following a replicative pathway

P element (Drosophila)Ac-Ds (maize)Tn3 and IS1 (E.coli)Tam3 (snapdragon)

Retroviral-like transposons

Directly repeated long terminal repeats (LTRs) at ends

Encodes reverse transcriptase and integrase -resembles retrovirus

Moves as DNA, but via an RNA intermediate produced by promoter in LTR

Copia (Drosophila)Ty1 (yeast)THE-1 (human)Bs1 (maize)

Nonretroviral retrotransposons

Poly A at 3’ end of RNA transcript; 5’ end is often truncated

Encodes reverse transcriptase; transposition is catalyzed by the RNA

Moves via an RNA intermediate that is often produced from a neighboring promoter

F element (Drosophila)L1 (human)Cin4 (maize)

Three Major Classes of Transposable Elements

AAATTT

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CLASS I: DNA-only transposons (1)

(2)

(3)

Bacteria has 3 classes

(1)

(3)

(2)

Examples:

Bacterial transposons

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1. Non-replicative mechanismSimple, cut-and-paste, no new DNA synthesis

2. Replicative mechanismMore complicated, copy-and-paste, with new DNA synthesis

Catalyzed by specialized recombination enzymes which recognize special DNA sequences (sites)

• Transposase (always)• Resolvase (sometimes)• Transposase inserts into target sites (<20 nt) on chromosome

Transposition is rare (~ once in 105 cell generations)

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Model for non-replicative transposition

Transposase cuts at the ends of the short inverted repeats and inserts transposon into new target DNA site

Insertion of transposon results in duplication of target sites

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A model for replicative transpostion

Involves DNA synthesis Tn3 transposes via

cointegrate formation

Tn3

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Transposase is a specialized recombination enzyme

Functions as a dimer, each monomer recognizes the same specific DNA sequence at the ends of the transposon

Dimerization of the subunits creates a DNA loop

Staggered cuts made on ends of target site

DNA Pol + Ligase

34Insertion of tranposon into new site & duplication of target sites

Recognition of target site

Mechanism of Transposition by Tn5

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Some viruses use transpositional site-specific recombination to move themselves into host chromosomesE.g. Bacteriophage Mu, retroviruses

Move via an RNA intermediate Use a reverse transcriptase and an integrase

(transposase)

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Retrovirus

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Integrase (transposase)

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Staggered cuts at target site

Insertion of retroviral DNA (or retrotransposons) into host genome

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CLASS III: Non-viral retrotransposons

Decendants of retroviral DNA (remnants of polyA tail)

no LTRs Occur as repetitive DNA

sequences (e.g. L1 element) Move via an endonuclease-

reverse transcriptase complex

Mechanism of retrotransposition

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2. An integrase (an endonuclease) generates staggered break at target site

3. A reverse transcriptase generates a cDNA copy of the retrotransposon

1. Retrotransposon is transcribed

E.g. LINE1

Petunia hybrida line W138 contains a disrupted rt locus for anthocyaninpigment production due to the.insertion of transposon dTph1. The mutation gives rise to a white flower.

Excision of Tph1 transposon in some cells during development restored pigment production (i.e. pink).

The pie-shaped pattern of cells reveals that the flower grows outward from a small number of cells in the center of the primordial flower head.

41Kroon, J et al 1994

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1. DNA-only transposons move by DNA breakage and joining

2. Retroviral-like retrotransposons also move by breakage and joining, but via an RNA intermediate

3. Non-retroviral retrotransposons move by making an RNA copy which acts as a direct template for a DNA target-primed reverse transcription event

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RecA-independent process Breakage and joining of DNA molecules occur

at a pair of specific sites involving very short homology (<50 bp)

Recombination is catalyzed by specific enzymes

Involved in the integration & excision of bacteriophage λ from the E.coli genome, and in the inversion of a DNA fragment

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attP and attB share a common core sequence recognized by Int & Xis

Site-specific recombination occurs between direct repeats - attP & attBor attL & attR, respectively

attP CAGCTTTTTTTATACTAAGTTGGTCGAAAAAAATATGATTCAAC

O site

(POP)

(Int, IHF)

attL attRattP

attB

POP’

BOB’

BOP’ POB’attL attR

(Xis)

(Integrase)

Integration and excision of λ into E. coli DNA

+ IHF

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“prophage”

Integration of λ

Alberts et al, Molecular Biology of the Cell 5th

Edition, p304-326

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