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

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

doi:10.1371/journal.pgen.1000356

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.

Highlights

Homologous recombination (HR) contributes to genome maintenance by repairing double-strand breaks (DSBs) and single-strand lesions
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

Summary

Introduction

Homologous recombination (HR) contributes to genome maintenance by repairing double-strand breaks (DSBs) and single-strand lesions. It accomplishes this by associating the damaged DNA with intact homologous sequences (reviewed in [1]). A major nuclease in this process is thought to be a complex containing three proteins: Mre, Rad and Nbs (called the MRN complex) (reviewed in [5]). Biochemical study demonstrated that Sae, a cofactor of the MRN complex, can process a single strand nick, and expand it [10]. The single-strand DNA generated adjacent to the DSB is coated with polymerized Rad, resulting in the formation of nucleoprotein filaments. The assembly of RAD51 at DNA damage sites is regulated by a number of RAD51 cofactors, including the tumor-suppressor gene BRCA1 (Breast Cancer Susceptibility Gene 1), BRCA2, and five

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