Recombination – read Chapter 11 New Topic Recombination – read Chapter 11

E. coli RecBCD Pathway of Homologous Recombination I Chi site:GCTGGTGG Crossover Non-crossover

Resolution of the Holliday intermediate

Binding of RecA to single-stranded DNA

Synapsis

RecA dependent synapsis

Figure 11-12 Figure 11-10

RecBCD Nicks DNA Near Chi (c) sites to Initiate Recombination Steps a and b of E. coli Rec BCD Pathway for Homologous Recombination Note: degredation pattern by RecB is affected by balance between ATP and Mg++. Here, ATP is present in excess.

Nicking of DNA by RecBCD Near to the Chi site Note: degredation pattern by RecB is affected by balance between ATP and Mg++. Here, ATP is present in excess.

Figure 11-5 Note: degredation pattern by RecB is affected by balance between ATP and Mg++. Here, Mg++ is present in excess.

Synthetic Holliday structure Figure 22.10

Assay for RuvA-RuvB Holliday junction complex All have ATPgS, except lane h

RuvAB

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.

Meiotic Recombination

Model for Meiotic Recombination in Yeast I probably Rad50 and 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.