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.
Highlights
In humans and mice, primary antibody (Ig) diversity is produced by V(D)J recombination, which is dependent on the RAG-1 and À2 proteins [1]
somatic hypermutation (SHM) and class switching absolutely depend on a mutator protein, activation-induced cytidine deaminase (AID or AICD), whose expression is restricted to activated B cells [3,4]
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
Summary
Primary antibody (Ig) diversity is produced by V(D)J recombination, which is dependent on the RAG-1 and À2 proteins [1]. In chicken Ig V genes, excision of AID-induced dU bases by UNG mostly leads to homology-directed gene conversion with WV genes by a process independent of translesion DNA repair, rather than to point mutation [21,24,25]. In yeast and vertebrate cell models, gene conversion is stimulated by the induction of a double-strand break (DSB), which produces the requisite free 39-ends [26,27]. This does not imply that DSBs are obligatory for gene conversion because free 39-ends are generated during DNA replication.
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