Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes
Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes
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DOI：
10.1371/journal.pgen.1000356
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发表时间：
2009-01-01
期刊：
影响因子：
4.5
通讯作者：
Takeda, Shunichi
中科院分区：
文献类型：
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作者：
Nakahara, Makoto;Sonoda, Eiichiro;Takeda, Shunichi
同源重组 (HR) 由 DNA 双链断裂 (DSB) 引发。然而，目前尚不清楚单链损伤是否也会启动基因组 DNA 中的 HR。鸡 B 淋巴细胞通过 HR（Ig 基因转换）和非模板超突变使其免疫球蛋白 (Ig) V 基因多样化。两种类型的 Ig V 多样化都是由 AID 依赖性脱碱基位点形成引发的。无碱基位点阻碍复制，导致单链缺口的形成。这些缺口可以通过容易出错的 DNA 聚合酶来填补，从而导致超突变。然而，尚不清楚这些单链缺口是否也可以启动 Ig 基因转换，而无需首先转换为 DSB。 Mre11-Rad50-Nbs1 (MRN) 复合物产生 39 个单链突出端，促进 DSB 诱导酵母中 HR 的启动。我们发现仅表达截短形式的 Nbs1 (Nbs1(p70)) 的 DT40 系表现出有缺陷的 HR 依赖性 DSB 修复，并且 Ig 基因转换的速率（尽管不是保真度）显着降低。有趣的是，这种有缺陷的基因转换通过大肠杆菌 SbcB（一种 3' 至 5' 单链特异性核酸外切酶）的过量生产恢复到野生型水平，而不影响 DSB 修复。相反，鸡 Exo1 的过度表达使 DSB 诱导的基因靶向效率提高了 10 倍以上，且对 Ig 基因转化没有影响。这些结果表明，Ig 基因转换可能是由单链缺口而不是 DSB 启动的，并且与 SbcB 一样，DT40 中的 MRN 复合物可能会将 AID 诱导的病变转化为适合触发 HR 的单链缺口。总之，Ig基因转换和超突变可能具有共同的底物——单链缺口。对 DT40 中两种 Ig V 多样化的遗传分析提供了一个独特的机会，可以深入了解由于 HR 和易错聚合酶在脱碱基位点复制阻断而产生的间隙填充的分子机制。
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 39 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.