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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?

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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?


Regulators of homologous recombination

donor repression

donor activation

point mutation

homology-directed repair


90

80

AcH3

AcH4

70

60

Fold Enrichment

50

40

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20

10

0

FlankV25 V24 V13 V5 V1 V1-V VR

Pseudogenes have an accessible chromatin state in DT40 cells


No IPTG 100 µM IPTG

15 µm

The experimental system


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GFP-LacI

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LacI-HP1

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Fold Enrichment

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2

1

0

AcH3 AcH4 AcH3 AcH4

V17

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
Effect of tethering HIRA

HIRA-LacI

GFP-LacI


Dt40 polylaco clones expressing histone h3 variants
DT40 PolyLacO clones expressinghistone H3 variants

H3.3-FLAG

H3.1-FLAG

C 1 2 3 4 1 2

FLAG

actin


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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
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

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direct repeats

or

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inverse repeats

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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)





Repair factors are limiting for gene conversion
Repair factors are limiting for gene conversion

Ellen Ordinario

Munehisa Yabuki


Competing repair pathways

Competing repair pathways

HDR w/

exogenous donor

Non-homologous

end joining

HDR w/

sister chromatid

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Smc5/6 …

Ku80 …

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Sequence

correction

Loss of sequence

Maintain sequence

information


Optimize hdr with an exogenous donor sequence
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


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