Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes

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Abstract
Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3′ single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1p70) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate—though not the fidelity—of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3′ to 5′ single-strand–specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate—single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases. An important class of chemotherapeutic drugs used in the treatment of cancer induces DNA damage that interferes with DNA replication. The resulting block to replication results in the formation of single-strand gaps in DNA. These gaps can be filled by specialized DNA polymerases, a process associated with the introduction of mutations or by recombination with an undamaged segment of DNA with an identical or similar sequence. Our work shows that diversification of the antibody genes in the chicken B cell line DT40, which is initiated by localized replication-stalling DNA damage, proceeds by formation of a single-strand intermediate. These gaps are generated by the action of a specific nuclease complex, comprising the Mre11, Rad50, and Nbs1 proteins, which have previously been implicated in the initiation of homologous recombination from double-strand breaks. However, in this context, their dysfunction can be reversed by the expression of a bacterial single-strand–specific nuclease, SbcB. Antibody diversification in DT40 thus provides an excellent model for studying the process of replication-stalling DNA damage and will allow a more detailed understanding of the mechanisms underlying gap repair and cellular tolerance of chemotherapeutic agents.