e. coli recbcd pathway of homologous recombination i
DESCRIPTION
E. coli RecBCD Pathway of Homologous Recombination I. Heteroduplexes; Non-crossover recombinants. Crossover recombinants. Resolution of the Holliday intermediate. Binding of RecA to single-stranded DNA. Synapsis. RecA dependent synapsis. RecBCD Appears to Nick DNA Near Chi - PowerPoint PPT PresentationTRANSCRIPT
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E. coli RecBCD Pathway of Homologous Recombination I
Heteroduplexes; Non-crossover recombinants.Crossover recombinants
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Resolution of the Holliday intermediate
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Binding of RecA to single-stranded DNA
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Synapsis
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RecA dependent synapsis
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RecBCD Appears to Nick DNA Near Chi () sites to Initiate Recombination
Steps a and b of E. coli Rec BCD Pathway for Homologous Recombination
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Nicking of DNA by RecBCD Near to the Chi site
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Figure 22.10
Synthetic Holliday structure
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Assay for RuvA-RuvB Holliday junction complex
All have ATPS, except lane h
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RuvAB
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RuvC Binds to Holliday Junctions
Gel shifts performed under noncleavage conditions
Synthetic Holliday junction
Dunderdale et al., Nature 354: 506-510, 1991
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RuvC Resolves Holliday Junctions
Gel shifts performed under cleavage conditions
Dunderdale et al., Nature 354: 506-510, 1991
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Properties of RuvC
•RuvC is a dimer and has two active sites.
•RuvC is thought to act on Holliday junctions already bound by RuvA and RuvB.
•Reason for RuvA/B requirement is that branch migration is required for resolution.
•RuvC cuts preferentially at 5’ (A/T)TT↓(G/C) 3’.
•Presumably branch migration is required to reach the preferred sequence for RuvC cutting.
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Meiotic Recombination
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Model for Meiotic Recombination in Yeast I
probably Rad50and Mre11
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Model for Meiotic Recombination in Yeast II
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Spo11 in Yeast makes double-stranded DNA breaks (DSBs)
•rad50S mutants accumulate DSBs and are a rich source of the protein that binds to DSBs
•Kleckner and colleagues isolated Spo11 from rad50S mutants
•Spo11 binds specifically to DSBs
•Cleavage to form a DSB occurs by a transesterification reaction in which attacking group is Tyr residue of Spo11
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Model for Participation of Spo11 in DSB Formation
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Figure 2. Location and amount of meiotic DSBs on chromosome III.