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E. coli RecBCD Pathway of Homologous Recombination I

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

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  1. E. coli RecBCD Pathway of Homologous Recombination I Heteroduplexes; Non-crossover recombinants. Crossover recombinants

  2. Resolution of the Holliday intermediate

  3. Binding of RecA to single-stranded DNA

  4. Synapsis

  5. RecA dependent synapsis

  6. 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

  7. Nicking of DNA by RecBCD Near to the Chi site

  8. Synthetic Holliday structure Figure 22.10

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

  10. RuvAB

  11. RuvC Binds to Holliday Junctions Synthetic Holliday junction Gel shifts performed under noncleavage conditions Dunderdale et al., Nature 354: 506-510, 1991

  12. RuvC Resolves Holliday Junctions Gel shifts performed under cleavage conditions Dunderdale et al., Nature 354: 506-510, 1991

  13. 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.

  14. Meiotic Recombination

  15. Model for Meiotic Recombination in Yeast I probably Rad50 and Mre11

  16. Model for Meiotic Recombination in Yeast II

  17. 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

  18. Model for Participation of Spo11 in DSB Formation

  19. Figure 2. Location and amount of meiotic DSBs on chromosome III.

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