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The chicken light chain locus: a model of homology-directed repair of SSBs?. V. V. V. V. C. V. J. V. V. V. V. C. V. J. Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation. V. V. V. V. C. V. J.
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The chicken light chain locus:a model of homology-directed repair of SSBs? V V V V C V J V V V V C V J Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation
V V V V C V J • 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?
Regulators of homologous recombination donor repression donor activation point mutation homology-directed repair
90 80 AcH3 AcH4 70 60 Fold Enrichment 50 40 30 20 10 0 FlankV25 V24 V13 V5 V1 V1-V VR Pseudogenes have an accessible chromatin state in DT40 cells
No IPTG 100 µM IPTG 15 µm The experimental system
10 GFP-LacI 9 LacI-HP1 8 7 6 Fold Enrichment 5 4 3 2 1 0 AcH3 AcH4 AcH3 AcH4 V17 pol ChIP analysis of LacI-HP1 effects on chromatin
IgM loss: a measure of mutation time in culture GFP-LacI LacI-HP1 Homology-directed events 77% 3% Nontemplated 23% 97%
VP16-LacI: Tethering an activator GFP-LacI VP16-LacI AcH4 IgG AcH4 IgG Ova V17 Enrichment: 4.5 12.4
H3 variants and HIRA Hake and Allis, PNAS (2006) Polo and Almouzni (2004)
Effect of tethering HIRA HIRA-LacI GFP-LacI
DT40 PolyLacO clones expressinghistone H3 variants H3.3-FLAG H3.1-FLAG C 1 2 3 4 1 2 FLAG actin
V V V V C V J • 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?
Recombination potential of SSBs versus DSBs I-SceI / AniI P T Puro eGFP eGFP • 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
Avoiding aberrant recombination at light chain locus C V J V V V V V V C V J V V V + direct repeats or V C V J V V V inverse repeats V V V
Aberrant recombination at light chain locus can occur by crossovers donor recipient SSBs rarely progress to Holliday intermediate (1) Holliday junctions are rarely resolved to crossovers (2)
RuvC resolvase: a possible diagnostic agent gene conversion crossover
Repair factors are limiting for gene conversion Ellen Ordinario Munehisa Yabuki
Competing repair pathways HDR w/ exogenous donor Non-homologous end joining HDR w/ sister chromatid xx xx xx xx xx xx Smc5/6 … Ku80 … x xx xx xx xx Sequence correction Loss of sequence Maintain sequence information
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