1. General homologous recombination 2.

Site-specific recombination

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
2

Genetic exchange takes place between 2 pieces of homologous DNA sequences May be intra- or intermolecular events

Heteroduplex formation at the site of crossover No alteration of nucleotide sequences at the site of exchange New recombinant DNA molecules are produced

Single-strand invasion model (MesselsonRadding model)

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

Single stranded DNA, coated by RecA, invades homologous duplex

Holliday junction

Resolution & Ligation

Double strand break model

1. Limited degradation at doublestrand break by a 53 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

Involvement of homologous recombination in repair of ds breaks

binds to single-stranded DNA (produced from nicks, ds breaks or gaps) mediates ATP-dependent strand invasion and exchange catalyzes branch migration 352 a.a.
Spooling in Spooling out

6 RecA monomers per turn

10

RecA is a long filamentous multisubunit protein complex that coats DNA in vivo
11

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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13

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helicase

35 & 53 exonucleases

ss DNA is coated by RecA for homologous recombination

15

~1000 chi sites (5'-GCTGGTGG-3) are present on the E. coli chromosome Chi sites are hotspots for general recombination

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RuvA
22 kD protein which binds to RuvB and Holliday junctions

RuvB
37 kD DNA-dep ATPase which drives branch migration

RuvC
19 kD nuclease which resolves Holliday structures (resolvase)

DNA ligase
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18

RuvA and RuvB recognize H-J and promotes branch migration

RuvC cleaves DNA strands at the H-J

Ligase seals the nicked strands

19

Gene conversion is non-reciprocal exchange Only small sections of DNA or part of a gene undergoes gene conversion

RecA mediated single-strand invasion

Heteroduplex DNA is formed

20

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

21

Homologous recombination pathways are shared by DNA repair mechanisms

Homologous strand exchange initiated by double strand break
3 5 3 5 5 5 3 3

Occurs predominantly in mitotic cells

3 5 3 5 5 3 5 3

Occurs predominantly in meiotic cells

22

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
23

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

25

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Three Major Classes of Transposable Elements

Class description and structure DNA-only transposons
Encodes transposase Short inverted repeats at each end Moves as DNA, either excising or following a replicative pathway

Genes in complete element

Mode of movement Examples

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
AAA TTT

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)

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CLASS I: DNA-only transposons

Bacteria has 3 classes
(2) (3) (1)

Examples:
(1) (3) (2)

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Bacterial transposons

29

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

2. Replicative mechanism
More 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 nonreplicative 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

33

Mechanism of Transposition by Tn5

Recognition of target site

Insertion of tranposon into new site & duplication of target sites

34

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

Move via an RNA intermediate Use a reverse transcriptase and an integrase (transposase)

35

Retrovirus

36

Integrase (transposase)

37

Insertion of retroviral DNA (or retrotransposons) into host genome

Staggered cuts at target site

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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 endonucleasereverse transcriptase complex

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Mechanism of retrotransposition
1. Retrotransposon is transcribed An integrase (an endonuclease) generates staggered break at target site A reverse transcriptase generates a cDNA copy of the retrotransposon

2.

3.

E.g. LINE1
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Petunia hybrida line W138 contains a disrupted rt locus for anthocyanin pigment 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. Kroon, 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 from the E.coli genome, and in the inversion of a DNA fragment
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Integration and excision of into E. coli DNA

POP

+ IHF (Integrase) (Int, IHF) (Xis)

attL attL
BOP

attP

attR attR

POB

BOB

attB

attP and attB share a common core sequence recognized by Int & Xis Site-specific recombination occurs between direct repeats - attP & attB or attL & attR, respectively O site attP CAGCTTTTTTTATACTAAGTTG (POP) GTCGAAAAAAATATGATTCAAC
46

Integration of

prophage
47

Alberts et al, Molecular Biology of the Cell 5th Edition, p304-326

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