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。导致这些突变的分子机制尚不清楚。证据表明，这一过程涉及靶向链断裂部位的DNA修复事件。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。其中一个或多个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)当复制未损坏的DNA时，聚合酶Zeta以5×10-4/bp的频率产生错误。在与R.Wood的合作中，我们破坏了聚合酶的催化亚单位，并产生了基因缺陷小鼠。然而，这些小鼠在怀孕中期死亡，这表明这种酶对胚胎发育至关重要。(B)聚合酶ETA在未损伤的DNA上以3×10~(-2)/bp的频率合成错误。在患有变异型干皮病的人中，这种酶是有缺陷的。我们对三名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。聚合酶ETA缺陷克隆在A和T的突变比例降低了三倍，而在G和C的突变增加。值得注意的是，这种突变模式的变化与聚合酶在体外复制未受损DNA时的特异性一致。这一发现表明，聚合酶ETA是超突变中的A-T突变子，另一个聚合酶作用于G和C核苷酸。(C)聚合酶IOTA在未损坏的模板上以3×10-1/bp的总频率出错。在与R.Woodgate的合作中，我们研究了在超突变过程中可能形成的DNA底物上的聚合酶IOTA的特异性。当聚合酶填充DNA末端的模板时，其保真度比填充较长模板时低10倍。由于突变发生在链断裂附近的可变基因中，聚合酶可能在超突变过程中很好地发挥突变因子的作用。

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.