title: Genetic evidence for single-strand lesions initiating Nbs1-dependent homologous recombination in diversification of Ig v in chicken B lymphocytes.
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 (Nbs1(p70)) 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.
authors.affiliation: CREST Research Project, Japan Science and Technology Agency, Saitama, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakahara
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Sonoda
authors.forename: Kuniharu
authors.initials: K
authors.lastname: Nojima
authors.forename: Julian E
authors.initials: JE
authors.lastname: Sale
authors.forename: Katsuya
authors.initials: K
authors.lastname: Takenaka
authors.forename: Koji
authors.initials: K
authors.lastname: Kikuchi
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Taniguchi
authors.forename: Kyoko
authors.initials: K
authors.lastname: Nakamura
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Sumitomo
authors.forename: Ronan T
authors.initials: RT
authors.lastname: Bree
authors.forename: Noel F
authors.initials: NF
authors.lastname: Lowndes
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D045744:Cell Line, Tumor; D002645:Chickens; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005785:Gene Conversion; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
chemical_list: D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I