DNA Repair And Somatic Mutation In Antibody Variable Gen

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)聚合酶Zeta。缺乏这种酶的小鼠在怀孕中期死亡，这表明这种酶对胚胎发育至关重要。将干扰RNA序列引入突变细胞系中，观察突变频率的降低。(B)聚合酶ETA。这种聚合酶在患有色素性干皮病的人中是有缺陷的。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缺失克隆的A、T位突变比例降低，G、C位突变比例升高。这一发现表明，聚合酶ETA是高突变中的A-T突变子。我们正在使用酵母双杂交系统来测试与聚合酶相互作用的蛋白质，并看看它是否参与了重链类开关区域的突变。(C)聚合酶IOTA。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。我们目前正在研究聚合酶缺陷小鼠的突变模式。

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. Mice deficient for this enzyme die during mid-gestation, suggesting that the enzyme is critical for embryonic development. Interfering RNA to the sequence will be introduced into a mutating cell line to see the frequency of mutation is lowered. (b) Polymerase eta. This polymerase 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 decrease in the proportion of mutations at A and T with a concomitant rise of mutations at G and C. This finding implies that polymerase eta is an A-T mutator in hypermutation. We are using the yeast two-hybrid system to test for proteins that interact with the polymerase, and to see if it is involved in generating mutations in the heavy chain class switch region. (c) Polymerase iota. 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. We are currently studying the pattern of mutations in mice deficient for the polymerase.