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Site-specific recombination
Site-specific recombination
Require very short (<5 bp) sequence homology; Special site recognition RecA-independent but require specialized proteins E.g. transposition
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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
Holliday junction
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
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
35 & 53 exonucleases
~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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Gene conversion is non-reciprocal exchange Only small sections of DNA or part of a gene undergoes gene conversion
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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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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P element (Drosophila) Ac-Ds (maize) Tn3 and IS1 (E.coli) Tam3 (snapdragon)
Retroviral-like transposons
Directly repeated long terminal repeats (LTRs) at ends
Nonretroviral retrotransposons
AAA TTT
Moves via an RNA intermediate that is often produced from a neighboring promoter
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Examples:
(1) (3) (2)
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Bacterial transposons
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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
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Tn3
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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)
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Retrovirus
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Integrase (transposase)
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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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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
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Integration of
prophage
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