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突变作为对DNA损伤的全局SOS响应的一部分瞬时发生。这个过程中的关键参与者是UmuD2C蛋白，它与活化的RecA（RecA*）和Ssb一起，以前被认为使DNA聚合酶III全酶能够穿过复制阻断病变。最近，随着UmuD2C复合物具有内在的DNA聚合酶活性的惊人发现，在阐明Umu依赖的跨损伤DNA合成的生化机制方面迈出了重要的一步，因此被称为E. UmuD诱变蛋白在功能上是无活性的，直到它经历RecA介导的翻译后切割反应以产生UmuD。与结构相关的莱克萨蛋白（其作为自切割研究的范例）不同，实验表明UmuD切割主要通过分子间反应发生。使用嵌合蛋白，我们能够鉴定UmuD内有效分子间切割所需的残基。我们的数据表明，只有当UmuD与自身形成双聚体时，才发生分子间裂解。在过去的一年里，我们还确定和表征了几个Umu同系物。其中之一，称为HumD，在噬菌体P1基因组中发现。有趣的是，HumD编码的蛋白质大小和结构与较短但具有致突变活性的UmuD蛋白相似，而不是较大的无活性UmuD蛋白。我们的研究表明HumD可以在功能上替代UmuD并与E. coli UmuC.我们以前已经描述了S。酿酒酵母Rad30蛋白。最近其他人的研究表明Rad30编码DNA聚合酶h。我们已经鉴定了pol h的新的人类和小鼠同源物，我们称之为RAD 30 B。人RAD30 B位于染色体18q21.1上，在一个区域中，该区域通常与人类癌症的病因学有关。小鼠Rad30b的原位分析显示，像许多修复蛋白一样，它在睾丸中高度表达。