The chicken light chain locus:
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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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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

30

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


10

GFP-LacI

9

LacI-HP1

8

7

6

Fold Enrichment

5

4

3

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 events77% 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


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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

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)


RuvC resolvase: a possible diagnostic agent

gene conversion

crossover


Maizels lab


Homology-directed repair(HDR)

Rad51…


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

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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

  • 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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