Dna Repair And Somatic Mutation In Antibody Genes

编码部分免疫球蛋白分子的可变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。引入这些突变的分子机制尚不清楚。证据指向一个过程，涉及DNA修复事件在目标链断裂的位置。在脊椎动物细胞中，有许多最近发现的DNA聚合酶不准确地复制模板。其中一种或多种是在超突变过程中引入碱基变化的酶的潜在候选者。我们正在研究DNA聚合酶zeta， eta和iota在这一机制中的作用。(a)聚合酶zeta在复制未损伤DNA时产生5 X 10-4/bp的错误。与R. Wood合作，我们破坏了聚合酶的催化亚基，并制造了基因缺陷小鼠。然而，这些小鼠在妊娠中期死亡，这表明这种酶对胚胎发育至关重要。(b)聚合酶eta在未损伤DNA上合成错误的频率为3 × 10-2/bp。这种酶在患有色素性干皮病的人身上是有缺陷的。我们对3例患者的可变基因进行了测序，发现他们的高突变频率是正常的，但碱基变化的类型不同。缺乏聚合酶的克隆在a和T位点的突变比例下降了三倍，同时在G和c位点的突变增加。值得注意的是，这种突变模式的转变与聚合酶在体外复制未受损DNA时的特异性是一致的。这一发现表明聚合酶在超突变中是A-T突变，另一种聚合酶作用于G和C核苷酸。(c)聚合酶iota在未损坏的模板上出错的总频率为3 X 10-1/bp。在与R. Woodgate的合作中，我们研究了iota聚合酶在DNA底物上的特异性，这些底物可能是在超突变期间形成的。当聚合酶在DNA末端填充模板时，其保真度比填充较长的模板时低10倍。由于突变发生在靠近链断裂的可变基因中，聚合酶很可能在超突变期间发挥突变子的作用。

Somatic hypermutation of variable genes, which encode a portion of immunoglobulin molecules, occurs at a frequency that is a million times greater than mutation in other genes. The molecular mechanism that introduces these mutations is unknown. Evidence points to a process that involves DNA repair events at sites of targeted strand breaks. In vertebrate cells, there are many recently identified DNA polymerases that inaccurately copy templates. One or more of these are potential candidates for enzymes that introduce base changes during hypermutation. We are studying the roles of DNA polymerases zeta, eta, and iota in the mechanism. (a) Polymerase zeta creates errors at a frequency of 5 X 10-4/bp when copying undamaged DNA. In collaboration with R. Wood, we disrupted the catalytic subunit of the polymerase and made gene-deficient mice. However, the mice died during mid-gestation, suggesting that the enzyme is critical for embryonic development. (b) Polymerase eta synthesizes errors at a frequency of 3 X 10-2/bp on non-damaged DNA. The enzyme is defective in people with xeroderma pigmentosum variant disease. We sequenced variable genes from three patients and found that their frequency of hypermutation was normal, but the types of base changes were different. Polymerase eta-deficient clones had a three-fold decrease in the proportion of mutations at A and T with a concomitant rise of mutations at G and C. It is notable that this shift in mutation pattern is consistent with the specificity of the polymerase when copying non-damaged DNA in vitro. This finding implies that polymerase eta is an A-T mutator in hypermutation, and another polymerase acts at G and C nucleotides. (c) Polymerase iota makes errors at an overall frequency of 3 X 10-1/bp on undamaged templates. In collaboration with R. Woodgate, we have studied the specificity of polymerase iota on DNA substrates that might be formed during hypermutation. The fidelities of the polymerase are 10-fold lower when it fills a template at a DNA terminus compared to when it fills a longer template. Since mutations occur in variable genes near strand breaks, the polymerase may well function as a mutator during hypermutation.