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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 Presentation
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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 () sites to Initiate Recombination
Steps a and b of E. coli Rec BCD Pathway for Homologous Recombination
Nicking of DNA by RecBCD Near to the Chi site
Figure 22.10
Synthetic Holliday structure
Assay for RuvA-RuvB Holliday junction complex
All have ATPS, except lane h
RuvAB
RuvC Binds to Holliday Junctions
Gel shifts performed under noncleavage conditions
Synthetic Holliday junction
Dunderdale et al., Nature 354: 506-510, 1991
RuvC Resolves Holliday Junctions
Gel shifts performed under cleavage conditions
Dunderdale et al., Nature 354: 506-510, 1991
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.
Meiotic Recombination
Model for Meiotic Recombination in Yeast I
probably Rad50and Mre11
Model for Meiotic Recombination in Yeast II
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
Model for Participation of Spo11 in DSB Formation
Figure 2. Location and amount of meiotic DSBs on chromosome III.