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. 2011 Mar 7;10(3):338-43.
doi: 10.1016/j.dnarep.2010.12.005. Epub 2011 Jan 20.

Competition between PARP-1 and Ku70 control the decision between high-fidelity and mutagenic DNA repair

M N Paddock  1 , A T Bauman, R Higdon, E Kolker, S Takeda, A M Scharenberg
Affiliations

Competition between PARP-1 and Ku70 control the decision between high-fidelity and mutagenic DNA repair

M N Paddock et al. DNA Repair (Amst). .

Abstract

Affinity maturation of antibodies requires a unique process of targeted mutation that allows changes to accumulate in the antibody genes while the rest of the genome is protected from off-target mutations that can be oncogenic. This targeting requires that the same deamination event be repaired either by a mutagenic or a high-fidelity pathway depending on the genomic location VSports手机版. We have previously shown that the BRCT domain of the DNA-damage sensor PARP-1 is required for mutagenic repair occurring in the context of IgH and IgL diversification in the chicken B cell line DT40. Here we show that immunoprecipitation of the BRCT domain of PARP-1 pulls down Ku70 and the DNA-PK complex although the BRCT domain of PARP-1 does not bind DNA, suggesting that this interaction is not DNA dependent. Through sequencing the IgL variable region in PARP-1(-/-) cells that also lack Ku70 or Lig4, we show that Ku70 or Lig4 deficiency restores GCV to PARP-1(-/-) cells and conclude that the mechanism by which PARP-1 is promoting mutagenic repair is by inhibiting high-fidelity repair which would otherwise be mediated by Ku70 and Lig4. .

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Figures

Fig. 1
Fig. 1
PARP inhibitors impair survival in response to challenge with MMS, but do not impair gene conversion, while overexpression of the BRCT domain of PARP-1 has the opposite effect. (A) Survival of indicated cell lines in response to MMS induced damage. Error bars indicate the SEM of three experiments. (B) Frequency of gene conversion events in respective cell lines. Error bars indicate the SEM of 2–4 cell lines cultured independently. (C) Schematic of BRCT overexpression construct. (D) Survival of indicated cell lines in response to MMS induced damage. Error bars indicate the SEM of three experiments. (E) Frequency of gene conversion events in respective cell lines. Error bars indicate the SEM of 2–4 cell lines cultured independently. (F) Western blot showing expression of the BRCT overexpression construct in multiple independent DT40 cell lines which fluoresced green, stained with anti-FLAG (Sigma).
Fig. 2
Fig. 2
Proteins which bind to the BRCT domain of PARP-1 were identified by mass spectrometry, confirmed by immunoblot, and validated by KO cell lines. (A) Graphical representation of the number of spectral identifications of proteins which were enriched in the BRCT overexpressing (BRCT o/e) cell line and the parent cell lines as a control. Proteins were coimmunoprecipitated with the BRCT construct by anti-FLAG antibodies bound to sepharose beads. (B) Western blot to verify enrichment of proteins identified by mass spectrometry. The BRCT domain was immunoprecipitated with anti-FLAG antibody and proteins which coimmunoprecipitated were probed with antibody to Ku70. (C) Frequency of gene conversion events in Ku70−/− cell lines. Error bars indicate the SEM of 2–4 cell lines cultured independently. (D) Frequency of gene conversion events in Lig4−/− cell lines. Error bars indicate the SEM of 2–4 cell lines cultured independently.
Fig. 3
Fig. 3
A model for the roles of PARP-1, Ku70, and Lig4 in somatic hypermutation and gene conversion. Ku70 and Lig4 mediate high fidelity repair unless inhibited by PARP-1, which perpetuates the lesion and shunts to a mutagenic repair pathway.
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