BACTERIOPHAGE P22 ESSENTIAL RECOMBINATION FUNCTION

拟议研究的广泛、长期目标是了解 一般重组的分子机制。未来的生物系统 被检测的是噬菌体P22及其近亲噬菌体 兰布达。在这些原核系统中，一般的重组起着 在保护染色体不受损害方面发挥重要作用。很有可能 重组/修复的基本原理在噬菌体中与 在实验中较难获得的高等真核生物中。了解这些 反过来，原则在理解人类疾病方面也很重要，例如 癌症，其根本原因是DNA损伤。 提出了一种以生物化学为导向的体外方法，基于 早期基因活体研究的发现。实验将集中在 这些系统本身的活动，以及它们与 宿主细胞重组系统，尤其是RecBCD。具体目标 包括； 波长红介导重组子最适条件的确定 在体外系统中；通过省略来建立系统要求 实验；与P22重组系统类似的实验。 尝试通过分离体外反应途径来实现这一过程 在早期阶段形成的络合物或中间体，随后是加成 后期所需的组件；结构特征 体外重组的中间体和产物。 经遗传鉴定的红豆杉必需蛋白质组分的纯化 系统(如果除了已知的RecA和lambda之外还发现了任何系统 核酸外切酶+红贝塔(或P22‘S ABC-RecBCD+Erf+Arf)。 对大肠杆菌提取物进行分级以提纯任何未鉴定的 基本组件。 Arf蛋白的纯化和性质，特别是关于 对Erf和RecA DNA结合活性的影响。 尝试获得适合于生物合成的Erf和Redbeta蛋白晶体 结晶学研究.Erf蛋白的结构-功能研究 特别想了解羧基末端结构域的功能。 ABC1-RecBCD和ABC1-ABC2-RecBCD的纯化及性质 它们与RecBCD、ABC2-的生化活性比较 和GAM-RecBCD；ABC2-RecBCD的生化研究， 尤其是关于双链形成的结构的类型 以存在和不存在纯化的ERF蛋白和参与 与Erf发生链交换反应。 使用体内lambda RFLP分析来检验对 基于生化的P22重组系统的要求 ABC2修饰的RecBCD的研究 GAM修饰的宿主因子的特征)负责 在lambda RFLP重组实验中对重组的刺激。

The broad, long-term objective of the proposed research to understand the molecular mechanism of general recombination. The biological systems to be examined are those of bacteriophage P22 and its relative, bacteriophage lambda. In these prokaryotic systems, general recombination plays an important role in protection of the chromosome from damage. It is likely that the basic principles of recombination/repair are the same in phages as in experimentally less accessible higher eukaryotes. Understanding these principles, in turn, is important in understanding human diseases, such as cancer, in which the underlying cause is DNA damage. A biochemically-oriented, in vitro approach is proposed, based upon the findings of earlier genetic, in vivo studies. Experiments will focus on the activities of these systems themselves, and on their interactions with the host cells recombination system, particularly RecBCD. Specific aims include; Determination of optimum conditions for lambda Red-mediated recombination in an in vitro system; establishment of system requirements by omission experiments; similar experiments with the P22 recombination system. Attempting to stage the in vitro reaction pathway through the isolation of complexes or intermediates formed in earlier stages, followed by addition of components needed at later stages; structural characterization of intermediates and products of recombination in vitro. Purification of genetically identified essential protein components of the system (if any are found in addition to the already-known RecA and lambda exonuclease + Redbeta (or P22's Abc-RecBCD + Erf + Arf). Fractionation of E. coli extracts for the purification of any unidentified essential components. Purification and characterization of Arf protein, especially with regard to effects on the DNA-binding activities of Erf and RecA. Attempts to obtain crystals of Erf and Redbeta proteins suitable for crystallographic studies; structure-function studies of Erf protein, in particular to learn about the function of the carboxy-terminal domain. Purification and characterization of Abc1-RecBCD and Abc1-Abc2-RecBCD; comparison of their biochemical activities with those of RecBCD, Abc2- RecBCD, and Gam-RecBCD; studies of the biochemistry of Abc2-RecBCD, especially wit regard to the types of structures formed at double-stranded ends in the presence and absence of purified ERF protein, and participation with Erf in strand exchange reactions. Use of the in vivo lambda RFLP assay to test predictions for the requirements of the P22 recombination system based on the biochemical studies of Abc2-modified RecBCD. Characterization of the Gam-modified host factors) responsible for stimulation of recombination in the lambda RFLP recombination assay.