DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS

真核和原核细胞中的 DNA 复制、修复和诱变

• 批准号: 6290230
• 负责人: ROGER WOODGATE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/6290230
• 关键词: DNA binding protein; DNA damage; DNA repair; DNA replication; Escherichia coli; Xenopus oocyte; bacterial proteins; gene mutation; human tissue; molecular cloning; radiation genetics; tissue /cell culture; ultraviolet radiation

Damage-induced SOS mutagenesis occurs transiently as part of the global SOS response to DNA damage. Key participants in this process are the UmuD2C proteins which together with activated RecA (RecA*), and Ssb were previously thought to enable DNA polymerase III holoenzyme to traverse otherwise replication-blocking lesions. A major step towards elucidating the biochemical mechanism of Umu-dependent translesion DNA synthesis was recently achieved with the striking discovery that the UmuD2C complex possesses intrinsic DNA polymerase activity and as a consequence has been called E. coli pol V. The UmuD mutagenesis protein is functionally inactive until it undergoes a RecA-mediated posttranslational cleavage reaction to generate UmuD. Unlike the structurally related LexA protein (which serves as a paradigm for self-cleavage studies), experiments revealed that UmuD cleavage predominantly occurs via an intermolecular reaction Using chimeric proteins, we were able to identify residues within UmuD that are required for efficient intermolecular cleavage. Our data suggests that intermolecular cleavage occurs only when UmuD forms a filament-dimer with itself. Within the past year, we have also identified and characterized several Umu homologs. One of these, called HumD, is found on the bacteriophage P1 genome. Interestingly, HumD encodes a protein of similar size and structure to the shorter, but mutagenically active, UmuD protein and not the larger inactive UmuD protein. Our studies revealed that HumD can functionally substitute for UmuD and promote mutagenesis together with E. coli UmuC. We have previously characterized the S. cerevisiae Rad30 protein. Studies by others recently demonstrated that Rad30 encodes DNA polymerase h. We have identified novel human and mouse homologs of pol h which we have called RAD30B. Human RAD30B is located on chromosome 18q21.1 in a region often implicated in the etiology of human cancers. In situ analysis of mouse Rad30b revealed that like many repair proteins, it is highly expressed in the testis.

损伤诱导的SOS诱变是作为对DNA损伤的全局SOS响应的一部分瞬时发生的。此过程中的主要参与者是UMUD2C蛋白，该蛋白与活化的RECA（RECA*）一起，以前认为SSB可以使DNA聚合酶III Holoenzyme能够穿越，以遍历其他复制阻滞性病变。最近，通过引人注目的发现，umud2c复合物具有内在的DNA聚合酶活性，因此，最近实现了迈向依赖UMU依赖性跨性别DNA合成的生化机制的主要步骤，其结果称为E. coli pol v。与结构相关的Lexa蛋白（作为自切除研究的范式）不同，实验表明，UMUD裂解主要通过使用嵌合蛋白的分子间反应发生，我们能够鉴定出有效的umud中的残基，这是有效的，这是有效的细胞间裂解所必需的。我们的数据表明，只有在Umud与自身形成细丝二聚体时，发生分子间的裂解。在过去的一年中，我们还确定并表征了几种UMU同源物。其中之一称为humd，在噬菌体P1基因组上发现。有趣的是，Humd编码与短相似的蛋白质和结构相似的蛋白质，但具有诱变活性，UMUD蛋白质而不是较大的非活性UMUD蛋白质。我们的研究表明，款项可以在功能上替代UMUD，并与大肠杆菌一起促进诱变。我们先前已经表征了酿酒酵母Rad30蛋白。其他人最近的研究表明，RAD30编码DNA聚合酶h。我们已经确定了我们称为rad30b的新型人和小鼠同源物。 Human Rad30b位于通常与人类癌症病因相关的区域中的18Q21.1染色体上。对小鼠Rad30b的原位分析表明，与许多修复蛋白一样，它在睾丸中高度表达。