DNA-Dependent Protein Kinase Inhibits AID-Induced Antibody Gene Conversion

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Abstract
Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID)-dependent hypermutation of Ig V(D)J rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(D)J regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(D)J hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion. To generate highly specific antibodies in response to an immune challenge, the antibody genes in activated B cells mutate at a very high rate over a period of several days. The enzyme that initiates antibody gene mutation is activation-induced cytidine deaminase (AID), the first protein recognized to directly edit DNA genomes in vivo. AID induces point mutation of antibody V genes in all vertebrates, as well as transfer of short sequences from nonfunctional donor V genes to functional acceptor V genes (“gene conversion”) in birds and some other species. Whether or not the mechanism of AID-induced V gene mutation and gene conversion involves double-strand DNA breaks is controversial and potentially important because double-strand DNA breaks are known to promote cancer-associated gene translocations. We used genetic inactivation of a double-strand break repair protein (DNA-dependent protein kinase) in a chicken B cell line to indirectly test whether AID induces double-strand breaks in the antibody V genes. We conclude that physiological expression of AID causes the formation of double-strand DNA ends in antibody V genes, which appear to be prevented from participating in homologous recombination if they recruit DNA-dependent protein kinase.