DNA Mismatch Repair in Bacillus subtilis

描述（申请人提供）：DNA错配修复（MMR）是所有生物体避免突变的主要途径。人类MMR基因的缺陷与包括遗传性非息肉病性结直肠癌在内的癌症的发生直接相关。细菌和真核细胞MMR蛋白之间惊人的序列同源性表明，探索细菌MMR将促进我们对突变避免，基因组稳定性和癌症发展的调控的理解。枯草芽孢杆菌是一种成熟的蛋白质定位实验系统，将被用作MMR的模式生物，因为它像真核生物一样不采用dam指导的甲基化来进行DNA链识别。这项工作将表征的本地化的B。枯草杆菌MMR蛋白MutS和MutL与体内DNA错配。我将监测与绿色荧光蛋白（GFP）融合的野生型和突变型MutS的定位。我将研究MutS突变蛋白与细菌持续合成因子（DNA聚合酶III的β亚基）的相互作用，以及它们参与链辨别。其他实验将试图确定参与MMR的辅助蛋白。这项工作将揭示MMR在活细胞框架内的精心编排。

DESCRIPTION (provided by applicant): DNA mismatch repair (MMR) is the major pathway of mutation avoidance in all organisms. Defects in human MMR genes have been directly linked to the development of cancer including hereditary nonpolyposis colorectal cancer. The striking sequence homology between bacterial and eukaryotic MMR proteins indicates that exploring bacterial MMR will advance our understanding of the regulation of mutation avoidance, genome stability and cancer development. Bacillus subtilis, a well-developed system for protein localization experiments, will be used as a model organism for MMR because like eukaryotes it does not employ dam-directed methylation for DNA strand discrimination. This work will characterize the localization of the B. subtilis MMR proteins MutS and MutL to DNA mismatches in vivo. I will monitor the localization of wild type and mutant MutS fused to green fluorescent protein (GFP). I will examine the interaction of MutS mutant proteins with the bacterial processivity factor, the beta subunit of DNA polymerase III, and their participation in strand discrimination. Other experiments will seek to identify accessory proteins that participate in MMR. This work will reveal the careful orchestration of MMR within the framework of a living cell.