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.