1. Recombination – read Chapter 11
New Topic
Recombination – read Chapter 11

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

3. Resolution of the Holliday intermediate

4. Binding of RecA to single-stranded DNA

5. Synapsis

6. RecA dependent synapsis

7. Figure 11-12
Figure 11-10

8. 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.

9. 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.

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

11. Synthetic Holliday structure
Figure 22.10

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

13. RuvAB

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

15. RuvC Resolves Holliday Junctions
Gel shifts performed under cleavage conditions
Dunderdale et al., Nature 354: , 1991

16. 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.

17. Meiotic Recombination

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

19. Model for Meiotic Recombination in Yeast II

20. 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

21. Model for Participation of Spo11 in DSB Formation

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