DNA Repair And Somatic Mutation In Antibody Variable Gen

免疫球蛋白基因的体细胞超突变发生的频率是其他基因突变的一百万倍。在恒定基因之前，在可变基因和开关区都发现了突变。引入这些突变的分子机制正在被深入研究。当激活诱导胞苷脱氨酶(AID)蛋白将DNA中的胞嘧啶脱氨为尿嘧啶时，就会启动超突变，从而导致C：G突变。然而，在B淋巴细胞中，所有四个碱基的替换发生在相似的水平，这表明需要其他蛋白质来产生A：T碱基对的突变。我们正在研究DNA聚合酶如何参与这一过程。聚合酶ETA显然通过引起A：T碱基对的替换参与了这一过程，而聚合酶IOTA的作用存在争议。为了在基因定义的背景下检查聚合酶iota，我们将129个无义突变回交到C57BL/6菌株，历时六代。在这些小鼠和聚合酶ETA和IOTA双重缺乏的同源小鼠中，研究了类开关重组和超突变。这两种聚合酶的缺失不影响IgG1的产生，表明这些酶不参与开关重组。Poli-/-F6小鼠与C57BL/6小鼠在可变基因上有相同类型的核苷酸替换，聚合酶ETA和IOTA双重缺陷的小鼠与Polh-/-小鼠具有相同的突变谱。因此，即使在没有聚合酶ETA的情况下，聚合酶IOTA对突变谱也没有贡献。

Somatic hypermutation of immunoglobulin genes occurs at a frequency that is a million times greater than mutation in other genes. Mutations are found in both variable genes and switch regions before constant genes. The molecular mechanism that introduces these mutations is intensely being studied. Hypermutation is initiated when the activation-induced cytidine deaminase (AID) protein deaminates cytosine in DNA to uracil, which causes C:G mutations. However, in B lymphocytes, substitutions of all four bases occur at similar levels, indicating that other proteins are required to generate mutations of A:T base pairs. We are studying how DNA polymerases are involved in the process. Polymerase eta is clearly involved in the process by causing substitutions of A:T basepairs, whereas polymerase iota has a controversial role. To examine polymerase iota in a genetically defined background, we backcrossed the 129 nonsense mutation to the C57BL/6 strain for six generations. Class switch recombination and hypermutation were studied in these mice and in congenic mice doubly deficient for both polymerases eta and iota. The absence of both polymerases did not affect production of IgG1, indicating that these enzymes are not involved in switch recombination. Poli-/-F6 mice had the same types of nucleotide substitutions in variable genes as their C57BL/6 counterparts, and mice doubly deficient for polymerases eta and iota had the same mutational spectrum as Polh-/- mice. Thus, polymerase iota did not contribute to the mutational spectra, even in the absence of polymerase eta.