The chicken light chain locus: a model of homology-directed repair of SSBs? VV VJ C VJ C Chicken...
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Transcript of The chicken light chain locus: a model of homology-directed repair of SSBs? VV VJ C VJ C Chicken...
![Page 1: The chicken light chain locus: a model of homology-directed repair of SSBs? VV VJ C VJ C Chicken pre-B cell line DT4O: carries out constitutive templated.](https://reader035.fdocuments.net/reader035/viewer/2022070418/5697bfaf1a28abf838c9d2ff/html5/thumbnails/1.jpg)
The chicken light chain locus:a model of homology-directed repair of SSBs?
V V J C
V J C
Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation
V V V
V V V V
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V V J CV 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?
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point mutation homology-directed repair
donor activationdonor repression
Regulators of homologous recombination
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Pseudogenes have an accessible chromatin state in DT40 cells
Flank V25 V24 V13 V5 V1 V1-V VR 0
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AcH3AcH4
Fold
En
rich
men
t
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No IPTG 100 µM IPTG
15 µm
The experimental system
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ChIP analysis of LacI-HP1 effects on chromatin
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AcH3 AcH4 AcH3 AcH4
V17 pol
GFP-LacI LacI-HP1
Fold
En
rich
men
t
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IgM loss: a measure of mutation
tim
e in
cu
ltu
re
GFP-LacI LacI-HP1Homology-directed events 77% 3% Nontemplated 23% 97%
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AcH4 IgG AcH4 IgG
V17
Ova
Enrichment: 4.5 12.4
GFP-LacI VP16-LacI
VP16-LacI: Tethering an activator
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H3 variants and HIRA
Hake and Allis, PNAS (2006) Polo and Almouzni (2004)
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Effect of tethering HIRA
GFP-LacI HIRA-LacI
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DT40 PolyLacO clones expressinghistone H3 variants
FLAG
actin
C 1 2 3 4 1 2
H3.1-FLAG H3.3-FLAG
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V V J CV 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?
![Page 13: The chicken light chain locus: a model of homology-directed repair of SSBs? VV VJ C VJ C Chicken pre-B cell line DT4O: carries out constitutive templated.](https://reader035.fdocuments.net/reader035/viewer/2022070418/5697bfaf1a28abf838c9d2ff/html5/thumbnails/13.jpg)
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
PuroeGFP eGFP
I-SceI / AniI
P T
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Avoiding aberrant recombination at light chain locus
V V J CV V V
V J CV V V
V
+direct repeats
or
VV
V
V J CV V
V V
V inverse repeats
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donor recipient
Aberrant recombination at light chain locuscan occur by crossovers
(1) SSBs rarely progress toHolliday intermediate
(2) Holliday junctions are rarelyresolved to crossovers
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RuvC resolvase: a possible diagnostic agent
crossover
gene conversion
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Maizels lab
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Homology-directed repair(HDR)
Rad51…
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Repair factors are limiting for gene conversion
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Munehisa Yabuki Ellen Ordinario
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Competing repair pathways
Ku80 …
Non-homologousend joining
Loss of sequence
HDR w/sister chromatid
Smc5/6 …
Maintain sequenceinformation
HDR w/exogenous donor
Sequencecorrection
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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