Regulating the Choice Between High-Fidelity and Mutagenic DNA Repair in Hypermutating B Cells

The process of affinity maturation allows generation of antibodies with increased affinity to a wider variety of antigens than would be possible if antibodies were limited to the sequences encoded by the germ line. Affinity maturation of antibodies requires a unique process of targeted mutation which allows changes to accumulate in the antibody genes and requires that the rest of the genome be protected from off-target mutations that can be oncogenic. To protect the genome from off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown. We investigated the role of the DNA damage sensor poly(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1 DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. We further performed functional analyses comparing the role of engineered PARP-1 variants in high- fidelity repair of DNA damage and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification. We further show that the BRCT domain of PARP-1 is able to coimmunoprecipitate Ku70 and the DNA-PK complex through a DNA- independent interaction. 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.