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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

The chicken light chain locus:a model of homology-directed repair of SSBs?

V

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J

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Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation


The chicken light chain locus a model of homology directed repair of ssbs

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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?


The chicken light chain locus a model of homology directed repair of ssbs

Regulators of homologous recombination

donor repression

donor activation

point mutation

homology-directed repair


The chicken light chain locus a model of homology directed repair of ssbs

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


The chicken light chain locus a model of homology directed repair of ssbs

No IPTG 100 µM IPTG

15 µm

The experimental system


The chicken light chain locus a model of homology directed repair of ssbs

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


The chicken light chain locus a model of homology directed repair of ssbs

IgM loss: a measure of mutation

time in culture

GFP-LacI LacI-HP1

Homology-directed events77% 3%

Nontemplated 23% 97%


The chicken light chain locus a model of homology directed repair of ssbs

VP16-LacI: Tethering an activator

GFP-LacI VP16-LacI

AcH4 IgG AcH4 IgG

Ova

V17

Enrichment: 4.5 12.4


The chicken light chain locus a model of homology directed repair of ssbs

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


The chicken light chain locus a model of homology directed repair of ssbs

V

V

V

V

C

V

J

  • 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


The chicken light chain locus a model of homology directed repair of ssbs

Avoiding aberrant recombination at light chain locus

C

V

J

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V

V

V

V

V

C

V

J

V

V

V

+

direct repeats

or

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C

V

J

V

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V

inverse repeats

V

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V


The chicken light chain locus a model of homology directed repair of ssbs

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)


The chicken light chain locus a model of homology directed repair of ssbs

RuvC resolvase: a possible diagnostic agent

gene conversion

crossover


The chicken light chain locus a model of homology directed repair of ssbs

Maizels lab


Homology directed repair hdr

Homology-directed repair(HDR)

Rad51…


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

xx

xx

xx

xx

xx

xx

Smc5/6 …

Ku80 …

x

xx

xx

xx

xx

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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