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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
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E. coli RecBCD Pathway of Homologous Recombination I Heteroduplexes; Non-crossover recombinants. Crossover recombinants
RecBCD Appears to Nick DNA Near Chi (c) sites to Initiate Recombination Steps a and b of E. coli Rec BCD Pathway for Homologous Recombination
Synthetic Holliday structure Figure 22.10
Assay for RuvA-RuvB Holliday junction complex All have ATPgS, except lane h
RuvC Binds to Holliday Junctions Synthetic Holliday junction Gel shifts performed under noncleavage conditions 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.
Model for Meiotic Recombination in Yeast I probably Rad50 and Mre11
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
Figure 2. Location and amount of meiotic DSBs on chromosome III.