Ordinario Ellen C, Yabuki Munehisa, Larson Ryan P, Maizels Nancy
Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA.
J Immunol. 2009 Oct 1;183(7):4545-53. doi: 10.4049/jimmunol.0900673. Epub 2009 Sep 11.
Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G(1) phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G(1) phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-lambda(R) cells, in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that both the repair polymerase Poleta and the multifunctional factor MRE11/RAD50/NBS1 localize to lambda(R), and that lambda(R)/Poleta colocalizations occur predominately in G(1) phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of gamma-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G(1) phase. AID deaminates ssDNA, and restriction of mutagenesis to G(1) phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.
重排的Ig V区在体细胞高频突变和基因转换的相关途径中，通过激活诱导的胞苷脱氨酶（AID）启动序列多样化，以产生非模板化或模板化突变。在鸡DT40 B细胞中，基因转换通常占主导地位，产生由相邻假V区作为模板的突变，但基因转换受损会使诱变切换到非模板化途径。我们最近表明，激活因子E2A以顺式作用促进多样化，并且细胞周期的G1期是E2A作用的关键窗口。通过对稳定转染AID-黄色荧光蛋白的细胞进行单细胞成像，我们现在证明AID-黄色荧光蛋白可以在G1期稳定地定位于细胞核，但在细胞周期后期会经历泛素依赖性蛋白水解。通过对DT40聚合乳糖操纵子-λ（R）细胞进行成像，其中聚合乳糖操纵子标记重排的λ（R）基因，我们表明修复聚合酶Poleta和多功能因子MRE11/RAD50/NBS1都定位于λ（R），并且λ（R）/Poleta共定位主要发生在G1期，此时它们反映了AID启动损伤的修复。我们没有发现γ-H2AX（双链断裂的标志物磷酸化变体组蛋白）诱导的证据，因此Ig基因转换可能通过涉及DNA切口处模板化修复而非双链断裂的途径进行。这些结果导致了一个模型，其中Ig基因转换在G1期启动并完成或几乎完成。AID使单链DNA脱氨基，并且将诱变限制在G1期将有助于在S期发生DNA复制时保护基因组免受AID的脱靶攻击。
