The chicken light chain locus: a model of homology-directed repair of SSBs? VV VJ C VJ C Chicken...

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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AcH3AcH4

Fold

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rich

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No IPTG 100 µM IPTG

15 µm

The experimental system

ChIP analysis of LacI-HP1 effects on chromatin

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AcH3 AcH4 AcH3 AcH4

V17 pol

GFP-LacI LacI-HP1

Fold

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IgM loss: a measure of mutation

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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

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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?

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

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Avoiding aberrant recombination at light chain locus

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+direct repeats

or

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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

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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