ERROR PRONE REPAIR, MUTAGENESIS AND AGING

拟议的项目是我们实现长期目标的第一步， 也就是在分子水平上理解 衰老中的突变机制。 由于哺乳动物中的诱变机制 系统在很大程度上是模糊的，甚至在原核生物中，一些系统的机制 主要的诱变途径是未知的，我们建议开始通过研究 SOS诱变的分子机制（也称为易错修复）， 特别是在E.杆菌 对这方面的独特兴趣 这一过程源于一个令人惊讶的发现，即它是一种主动诱变， 需要诱导特定基因产物的过程，recA， umuC，umuD，也许更多。 我们的战略主要采用生物化学方法， 完整的体外复制系统，用于复制紫外线照射的 DNA，使用野生型和SOS诱导的细胞作为蛋白质来源， 并测定新生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， 底物将含有单一的嘧啶光二聚体（主要的UV 在特定部位的病变。 这些都将被用来直接研究的作用 的主要紫外线损伤的诱变在体内和体外。 纯化 复制系统，主要由A.科恩伯格和他的同事们 将在整个研究过程中使用。 其中包括复制 单链DNA从GM 13和G4到复制型（SS 导致RF反应）和噬菌体复制型I DNA的复制 Phi-X174转化为单链DNA（FR导致SS反应）。 详细 嘧啶旁路和终止的生化分析 光二聚体的DNA聚合酶III全酶，主要复制 E.大肠杆菌，将进行。 这些研究将是 然后试图阐明SOS的作用诱导蛋白 例如UV诱变中umuC和umuD。 理解E.大肠杆菌会给我们一些线索 研究哺乳动物中类似过程的起始指南 系统和开发的方法将应用于实际的 研究了果蝇的突变及其在衰老中的作用。

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.