1. The chicken light chain locus: a model of homology-directed repair of SSBs? VV VJ C VJ C Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation VV VV VV VV VV VV VV

2. VV VJ C VV VV VV Regulators of homology-directed repair after the DNA lesion –What makes an attractive donor? SSBs –What is the true recombination potential of nicks? –Are nicks blocks to aberrant recombination at the light chain locus?

3. point mutationhomology-directed repair donor activation donor repression Regulators of homologous recombination

4. Pseudogenes have an accessible chromatin state in DT40 cells

5. No IPTG 100 µM IPTG 15 µm The experimental system

6. ChIP analysis of LacI-HP1 effects on chromatin 0 1 2 3 4 5 6 7 8 9 10 AcH3 AcH4 AcH3 AcH4  V 17 pol  GFP-LacI LacI-HP1 Fold Enrichment

7. IgM loss: a measure of mutation time in culture GFP-LacI LacI-HP1 Homology-directed events77% 3% Nontemplated 23% 97%

8.  AcH4 IgG  V17 Ova Enrichment: 4.5 12.4 GFP-LacI VP16-LacI VP16-LacI: Tethering an activator

9. H3 variants and HIRA Hake and Allis, PNAS (2006) Polo and Almouzni (2004)

10. Effect of tethering HIRA GFP-LacI HIRA-LacI

11. DT40 PolyLacO clones expressing histone H3 variants  FLAG  actin C 1 2 3 4 1 2 H3.1-FLAG H3.3-FLAG

12. VV VJ C VV VV VV Regulators of homology-directed repair after the DNA lesion –What makes an attractive donor? SSBs –What is the true recombination potential of nicks? –Are nicks blocks to aberrant recombination at the light chain locus?

13. Recombination potential of SSBs versus DSBs  have stable clones in DT40, Ramos and Nalm6 cell lines  analysis of the DSB and nickase activities of wild-type and mutant AniI  constructing integrating retroviral vectors for delivery of endonucleases Puro eGFP I-SceI / AniI PT

14. Avoiding aberrant recombination at light chain locus VV VJ C VV VV VV VJ C VV VV VV VV + direct repeats or V VV VV VJ C VV VV VV V VV inverse repeats

15. donorrecipient Aberrant recombination at light chain locus can occur by crossovers (1) SSBs rarely progress to Holliday intermediate (2) Holliday junctions are rarely resolved to crossovers

16. RuvC resolvase: a possible diagnostic agent crossover gene conversion

17. Maizels lab

18. Homology-directed repair (HDR) Rad51…

19. Repair factors are limiting for gene conversion Munehisa Yabuki Ellen Ordinario

20. Competing repair pathways Ku80 …  Non-homologous end joining Loss of sequence HDR w/ sister chromatid Smc5/6 … Maintain sequence information HDR w/ exogenous donor Sequence correction xx x

21. Optimize HDR with an exogenous donor sequence Express factors that are limiting for HDR (e.g. NBS1) Knockdown non-homologous end joining factors (e.g. Ku80) Knockdown factors (e.g. SMC5) that promote HDR with the sister chromatid