Dna Repair And Somatic Mutation In Antibody Genes

抗体基因中的 DNA 修复和体细胞突变

• 批准号: 6530369
• 负责人: Vilhelm A Bohr
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6530369
• 关键词: DNA directed DNA polymerase; DNA repair; aging; developmental genetics; enzyme activity; gene mutation; gene targeting; genetically modified animals; human subject; human tissue; immunoglobulin genes; laboratory mouse; nucleic acid sequence; restriction endonucleases

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.

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。有证据表明，一个过程涉及目标链断裂位点的 DNA 修复事件。在脊椎动物细胞中，有许多最近发现的 DNA 聚合酶可以不准确地复制模板。其中一种或多种是在超突变期间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 在该机制中的作用。 (a) 复制未损坏的 DNA 时，聚合酶 zeta 会以 5 X 10-4/bp 的频率产生错误。我们与 R. Wood 合作，破坏了聚合酶的催化亚基，并培育出了基因缺陷小鼠。然而，小鼠在妊娠中期死亡，这表明该酶对于胚胎发育至关重要。 (b) 聚合酶 eta 在未受损 DNA 上以 3 X 10-2/bp 的频率合成错误。患有着色性干皮病变异性疾病的人体内的酶存在缺陷。我们对三名患者的可变基因进行了测序，发现他们的超突变频率正常，但碱基变化的类型不同。聚合酶eta缺陷克隆的A和T突变比例减少了三倍，同时G和C突变增加。值得注意的是，突变模式的这种转变与复制时聚合酶的特异性一致体外 DNA 未受损。这一发现意味着聚合酶eta是超突变中的A-T突变子，而另一种聚合酶作用于G和C核苷酸。 (c) 聚合酶 iota 在未损坏的模板上以 3 X 10-1/bp 的总体频率发生错误。我们与 R. Woodgate 合作，研究了超突变过程中可能形成的 DNA 底物上聚合酶 iota 的特异性。与填充较长模板时相比，在 DNA 末端填充模板时，聚合酶的保真度要低 10 倍。由于突变发生在链断裂附近的可变基因中，因此聚合酶在超突变期间可以很好地发挥突变子的作用。