The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

Abstract

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here 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. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) 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, while being dispensable for the high-fidelity base excision repair. 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.