Mismatch Repair in V region mutation and isotype switch

描述（由申请人提供）：对多种错配修复蛋白存在遗传缺陷的小鼠的研究强烈表明，错配修复（MMR）通过影响抗体可变区基因的体细胞超突变（SHM）和类开关重组（CSR），在抗体多样性的产生中起着重要作用。此外，MMR缺乏易患B细胞和T细胞恶性肿瘤。对小鼠MSH2和MSH6缺陷的研究已经得出假设，SHM和CSR可能发生在两个阶段：一个阶段依赖于AID，导致热点地区G和C的突变；另一个阶段依赖于MMR，导致所有碱基的突变，并不局限于热点基序。我建议通过检测外切酶1缺陷或表达MMR蛋白的突变使其结合ATP的能力失活的小鼠来验证这一假设并了解更多关于MMR的作用。缺乏MMR活性的小鼠的表型可能部分反映了B细胞产生更高亲和力或自身反应性抗体的阳性和阴性选择。因此，我也将研究错配修复的作用，并通过使每种已知的MMR蛋白失活，并在培养的抗体形成细胞中表达突变蛋白，剖析单个错配修复蛋白的影响，在培养的细胞中，突变抗体的阳性和阴性选择将不会发生。我将使用MMR修复缺陷细胞来寻找与SHM有关的其他蛋白质。这些研究将为SHM和CSR的生化机制以及MMR在B细胞恶性肿瘤和其他癌症易感性中的作用提供新的见解。

DESCRIPTION (provided by applicant): Studies in mice that are genetically defective in various mismatch repair proteins strongly suggest that mismatch repair (MMR) plays a major role in the generation of antibody diversity through its affect on somatic hypermutation (SHM) of antibody variable region genes and on class switch recombination (CSR). In addition, deficiencies in MMR predispose to B and T cell malignancies. Studies with of mice MSH2 and MSH6 deficiencies have lead to the hypothesis that SHM and perhaps CSR occur in two phases: one that is AID dependent and results in mutations in G and C in hotspots and a second that depends upon MMR and results in mutations in all bases and is not restricted to hot spot motifs. I propose to test this hypothesis and learn more about the role of MMR by examining mice that are defective in exonuclease 1 or are expressing MMR proteins that have mutations that inactivate their ability to bind ATP. It is possible that the phenotypes of mice lacking MMR activity is partly the reflection of positive and negative selection for B cells making higher affinity or self reactive antibodies. I will therefore also study the role of mismatch repair and dissect out the impact of individual mismatch repair protein by inactivating each of the known MMR proteins and expressing mutant proteins in antibody-forming cells in culture where the positive and negative selection of mutated antibodies will not occur. I will use the MMR repair deficient cells to search for additional proteins that are involve in SHM. These studies should provide new insights into the biochemical mechanisms of SHM and CSR and the role of MMR in predisposing to B cell malignancies and other cancers.