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The chicken light chain locus:a model of homology-directed repair of SSBs?
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Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation
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• 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?
point mutation homology-directed repair
donor activationdonor repression
Regulators of homologous recombination
Pseudogenes have an accessible chromatin state in DT40 cells
Flank V25 V24 V13 V5 V1 V1-V VR 0
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30
40
50
60
70
80
90
AcH3AcH4
Fold
En
rich
men
t
No IPTG 100 µM IPTG
15 µm
The experimental system
ChIP analysis of LacI-HP1 effects on chromatin
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10
AcH3 AcH4 AcH3 AcH4
V17 pol
GFP-LacI LacI-HP1
Fold
En
rich
men
t
IgM loss: a measure of mutation
tim
e in
cu
ltu
re
GFP-LacI LacI-HP1Homology-directed events 77% 3% Nontemplated 23% 97%
AcH4 IgG AcH4 IgG
V17
Ova
Enrichment: 4.5 12.4
GFP-LacI VP16-LacI
VP16-LacI: Tethering an activator
H3 variants and HIRA
Hake and Allis, PNAS (2006) Polo and Almouzni (2004)
Effect of tethering HIRA
GFP-LacI HIRA-LacI
DT40 PolyLacO clones expressinghistone H3 variants
FLAG
actin
C 1 2 3 4 1 2
H3.1-FLAG H3.3-FLAG
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?
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
Avoiding aberrant recombination at light chain locus
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V
+direct repeats
or
VV
V
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V inverse repeats
donor recipient
Aberrant recombination at light chain locuscan occur by crossovers
(1) SSBs rarely progress toHolliday intermediate
(2) Holliday junctions are rarelyresolved to crossovers
RuvC resolvase: a possible diagnostic agent
crossover
gene conversion
Maizels lab
Homology-directed repair(HDR)
Rad51…
Repair factors are limiting for gene conversion
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.QuickTime™ and a
TIFF (Uncompressed) decompressorare needed to see this picture.
Munehisa Yabuki Ellen Ordinario
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