DNA REPAIR AND SOMATIC MUTATION IN ANTIBODY VARIABLE GENES

抗体可变基因中的 DNA 修复和体细胞突变

• 批准号: 6431457
• 负责人: PATRICIA J GEARHART
• 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/6431457
• 关键词: DNA repair; gel electrophoresis; gene expression; gene mutation; genetically modified animals; hybridomas; laboratory mouse; molecular cloning; nucleic acid sequence; polymerase chain reaction; restriction endonucleases; southern blotting; ultraviolet radiation

Somatic hypermutation of variable (V) 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. The project has two major aims. The first goal was to determine which DNA polymerases are expressed in B lymphocytes undergoing mutation. DNA polymerases alpha, beta, delta, epsilon and zeta are expressed in the rapidly dividing cells from germinal centers, and they could all be candidates for the enzyme introducing mutations. However, polymerases alpha and epsilon are not expressed by B cells outside of germinal centers, suggesting that these cells are resting and are deficient for DNA repair that utilizes polymerase epsilon. In addition, disruption of polymerase zeta in a mouse model caused embryonic lethality, suggesting that it is essential for cell viability during mammalian embryonic development. The second goal was to analyze hypermutation in V genes from old and young humans to determine if the frequency or pattern of mutation changes with age. The frequency of mutation was identical in both groups, indicating that old humans have hypermutated antibodies with high affinity for antigens. The frequency of tandem mutations, which have been associated with defective DNA mismatch repair in mice, varied among individuals. Microsatellite variability in DNA, which is caused by impaired mismatch repair, was then measured, and there was a strong correlation between the frequency of tandem mutations and microsatellite alterations. The data suggest that tandem mutations in human variable genes are related to defective mismatch repair.

编码部分免疫球蛋白分子的可变(V)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。该项目有两个主要目标。第一个目标是确定在发生突变的B淋巴细胞中表达哪些DNA聚合酶。DNA聚合酶α、β、Delta、epsilon和Zeta在生发中心快速分裂的细胞中表达，它们都可能是酶诱导突变的候选者。然而，生发中心外的B细胞不表达聚合酶α和epsilon，这表明这些细胞处于休眠状态，缺乏利用聚合酶epsilon进行DNA修复的能力。此外，破坏小鼠模型中的聚合酶Zeta会导致胚胎死亡，这表明在哺乳动物胚胎发育过程中，聚合酶Zeta对细胞存活至关重要。第二个目标是分析老年人和年轻人的V基因的超突变，以确定突变的频率或模式是否会随着年龄的变化而变化。这两组人的突变频率是相同的，这表明老年人拥有对抗原具有高亲和力的超突变抗体。与小鼠DNA错配修复缺陷有关的串联突变的频率因个体而异。然后测量了DNA中由错配修复受损引起的微卫星变异性，发现串联突变的频率与微卫星变化之间存在很强的相关性。这些数据表明，人类可变基因的串联突变与错配修复缺陷有关。