Genetic evidence for single-strand lesions initiating Nbs1-dependent homologous recombination in diversification of Ig V in chicken B lymphocytes

Makoto Nakahara, Eiichiro Sonoda, Kuniharu Nojima, Julian E. Sale, Katsuya Takenaka, Koji Kikuchi, Yoshihito Taniguchi, Kyoko Nakamura, Yoshiki Sumitomo, Ronan T. Bree, Noel F. Lowndes, Shunichi Takeda

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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.

    Original languageEnglish (US)
    Article numbere1000356
    JournalPLoS genetics
    Volume5
    Issue number1
    DOIs
    StatePublished - Jan 1 2009

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    ASJC Scopus subject areas

    • Ecology, Evolution, Behavior and Systematics
    • Molecular Biology
    • Genetics
    • Genetics(clinical)
    • Cancer Research

    Cite this

    Nakahara, M., Sonoda, E., Nojima, K., Sale, J. E., Takenaka, K., Kikuchi, K., Taniguchi, Y., Nakamura, K., Sumitomo, Y., Bree, R. T., Lowndes, N. F., & Takeda, S. (2009). Genetic evidence for single-strand lesions initiating Nbs1-dependent homologous recombination in diversification of Ig V in chicken B lymphocytes. PLoS genetics, 5(1), [e1000356]. https://doi.org/10.1371/journal.pgen.1000356