ERROR PRONE REPAIR--MUTAGENESIS AND AGING

拟议的项目是迈向我们长期目标的第一步， 这是对分子水平上的作用的理解， 衰老中的诱变机制。由于哺乳动物的诱变机制 系统在很大程度上是晦涩难懂的，甚至在原核生物中，一些 主要的诱变途径尚不清楚，我们建议从研究 SOS突变的分子机制(也称为易错修复)， 尤其是在大肠杆菌中的紫外线诱变。对这件事的独特兴趣 这一过程源于一个令人惊讶的发现，即它是一种活跃的突变 需要诱导特定基因产物即recA的过程， UMUC，UMD，或许更多。 我们的战略，主要是采用生化方法，是利用 用于紫外线照射的复制的完整的体外复制系统 DNA，使用野生型和SOS诱导的细胞作为蛋白质来源， 以及检测新生的DNA长度或碱基变化的产生。我们 我还建议开发一种制备特殊DNA的方法 将包含单个嘧啶光二聚体(主要的UV 损伤)在特定位置。这些将被用来直接研究角色 体内和体外诱变中的主要紫外线损伤。纯净的 复制系统，主要由A.Kornberg和他的同事开发 将在整个研究过程中使用。其中将包括复制 从噬菌体M13和G4到复制形式(SS)的单链DNA 导致RF反应)和复制的I型噬菌体DNA Phi-x174与单链DNA结合(FR导致SS反应)。一个详细的 嘧啶的旁路和终止的生化分析 光二聚体由DNA聚合酶III全酶复制而成 大肠杆菌的聚合酶，将被进行。这些研究将是 然后试图阐明S.O.S诱导的蛋白质的作用 如uMUC和umuD在紫外线诱变中的作用。 了解大肠杆菌中容易出错的修复将给我们提供线索和一些 研究哺乳动物类似过程的起始指南 系统，所开发的方法将应用于实际 哺乳动物的突变及其在衰老中的作用的研究。

The proposed project represents the first step towards our long term goal, which is the understanding, on a molecular level, of the role of mutagenesis mechanisms in aging. Since mutagenesis mechanisms in mammalian systems are largely obscure, and even in prokaryotes the mechanisms of some major mutagenesis pathways are unknown, we propose to begin by studying the molecular mechanism of SOS-mutagenesis (also termed error-prone repair), and in particular UV-mutagenesis in E. coli. The unique interest in this process stems from the surprising finding that it is an active mutagenesis process which requires the induction of specific gene product i.e., recA, umuC, umuD and maybe more. Our strategy, adopting primarily a biochemical approach, is to utilize complete in vitro replication systems for the replication of UV-irradiated DNA, using both wild-type and SOS-induced cells as the proteins sources, and assay for nascent DNA length or the production of base changes. We also propose to develop a methodology for the preparation of special DNA substrates which will contain a single pyrimidine photodimer (the major UV lesion) at a specific site. These will be used to study directly the role of the major UV-lesion in mutagenesis in vivo and in vitro. Purified replication systems, developed primarily by A. Kornberg and his colleagues will be used throughout the study. These will include replication of single stranded DNA from phages M13 and G4 to the replicative form (SS leads to RF reaction) and replication of replicative form I DNA of phage Phi-X174 to single stranded DNA (FR leads to SS reaction). A detailed biochemical analysis of bypass of, and termination at pyrimidine photodimers by DNA polymerase III holoenzyme, the major replicative polymerase of E. coli, will be carried out. These studies are to be followed by an attempt to elucidate the role of S.O.S. induced proteins such as umuC and umuD in UV-mutagenesis. Understanding error-prone repair in E. coli will give us clues and some starting guidelines for investigating similar processes in mammalian systems, and the methodologies developed will be applied for the actual study of mutagenesis in mammlians, and its role in aging.