BACTERIOPHAGE P22 ESSENTIAL RECOMBINATION FUNCTION

噬菌体 P22 基本重组功能

• 批准号: 2190258
• 负责人: ANTHONY R. POTEETE
• 金额: $ 24.83万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190258
• 关键词: DNA binding protein; DNA repair; DNA replication; bacteriophage P22; bacteriophage lambda; exonuclease; genetic recombination; protein purification; restriction fragment length polymorphism; virus replication

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.

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 检测的是噬菌体P22及其相关噬菌体的那些 的λ 在这些原核系统中，一般重组起着重要作用。 在保护染色体免受损伤中起重要作用。 很可能 重组/修复的基本原理在大肠杆菌中是相同的， 在实验上不易接近的高等真核生物中。 了解这些 原则，反过来，在理解人类疾病，如 癌症，其根本原因是DNA损伤。 一个生物化学导向的，在体外的方法提出，基于 早期的遗传学和体内研究的结果。 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，特别是RecBCD。 具体目标 包括; λ Red介导的重组的最佳条件的确定 在体外系统中;通过省略确定系统要求 实验;用P22重组系统进行的类似实验。 试图通过分离的体外反应途径， 在早期阶段形成的络合物或中间体，然后加入 后期阶段所需的组件; 体外重组的中间体和产物。 纯化经遗传鉴定的大肠杆菌的必需蛋白质组分 系统（如果除了已知的RecA和lambda之外还发现任何 外切核酸酶+ Redbeta（或P22的Abc-RecBCD + Erf + Arf）。 E.大肠杆菌提取物，用于纯化任何未鉴定的 重要的组成部分。 Arf蛋白的纯化和表征，特别是关于 对Erf和RecA的DNA结合活性的影响。 尝试获得适合于制备的Erf和Redbeta蛋白的晶体。 晶体学研究; Erf蛋白的结构-功能研究， 特别是了解羧基末端结构域的功能。 Abc 1-RecBCD和Abc 1-Abc 2-RecBCD的纯化和性质研究 与RecBCD、Abc 2- RecBCD和Gam-RecBCD; Abc 2-RecBCD的生物化学研究， 特别是考虑到在双链处形成的结构类型 在存在和不存在纯化的ERF蛋白的情况下结束， 与Erf在链交换反应中。 使用体内Lambda RFLP测定来测试对以下的预测： 基于生物化学的P22重组系统的要求 Abc 2修饰的RecBCD的研究。 Gam修饰的宿主因子的表征） 在λ RFLP重组测定中刺激重组。