Recombination and fork progression in bacteriophage T4

描述（由申请人提供）：同源重组是DNA代谢中的基本事件。重组在产生遗传多样性中的作用早已被人们所认识，现在人们知道重组对DNA修复和拯救停滞的复制叉至关重要。高等生物中这些修复机制的缺陷导致突变的积累，最终导致癌症，因此拟议的研究与人类疾病直接相关。我们有兴趣在结构水平上了解重组的潜在机制，并建议在一个非常简单的，特征良好的生物体，即噬菌体T4中研究它们。T4是这些研究的理想系统，因为它依赖于重组依赖性复制或RDR和有效的复制叉进展，以在大肠杆菌感染周期中产生所需水平的DNA。 将研究七种T4蛋白，UvsX、UvsY、UvsW、UvsW.1、Dda、gp 32和内切核酸酶VII。重组蛋白三联体UvsX、UvsY和UvsW介导同源重组反应的核心，并且分别与真核蛋白Rad 51、Rad 52和Rad 54相关。UvsW和Dda是转位和/或解旋分支核酸结构的解旋酶，并且在重组和复制叉进展中具有重要作用。已知Bloom和Werner等解旋酶的缺陷会导致人类癌症，并且有证据表明UvsW和Dda可能与这些分子的功能非常相似。UvsW.1是一个以前未知的T4蛋白，我们已经确定，在重组的推定作用。gp 32是T4单链DNA结合蛋白，已知其在T4 DNA代谢的许多方面具有关键作用。最后，核酸内切酶VII分解霍利迪连接以完成同源重组反应。这七种蛋白质的机制和相互作用将在分子水平上通过协调的方法进行研究，包括X射线晶体学研究它们的结构，体外方法研究它们的个体功能和相互作用，以及体内方法了解它们的生物学作用。该项目已经获得了相当多的初步数据，包括晶体和NMR结构，重要的初步晶体，纯化的蛋白质，生化活性的演示，以及基于T4突变体分析的体内功能。 公共卫生相关性：该项目的重点是在所有生命形式中运作的基本DNA代谢事件，同源重组（HR）。传统上与遗传多样性的传播有关，HR现在被认为是准确和快速修复各种形式DNA损伤的主要机制。许多形式的癌症，特别是乳腺癌，都与HR机制的缺陷有关。 HR的中心事件是DNA链的配对，同源性的搜索和同源DNA片段的交换。虽然介导这一显着过程的蛋白质被很好地表征，但在结构水平上的实际机制还不清楚。该项目的目标是研究简单噬菌体T4系统中的HR，了解T4蛋白质组分如何协调反应，并研究HR如何介导T4和高等生物中的DNA修复。该项目包括结构，遗传学和生物化学技术，共同研究这些重要的生物学问题。

DESCRIPTION (provided by applicant): Homologous recombination is a fundamental event in DNA metabolism. Long recognized for its role in generating genetic diversity, recombination is now known to be crucial for DNA repair and the rescue of stalled replication forks. Defects in these repair mechanisms in higher organisms lead to the accumulation of mutations that eventually result in cancer, and the proposed studies are therefore directly relevant to human disease. We are interested in understanding the underlying mechanisms of recombination at the structural level, and propose to study them in a very simple, well characterized organism, namely bacteriophage T4. T4 is an ideal system for these studies because it relies on recombination-dependent replication or RDR and efficient replication fork progression to generate the required levels of DNA during its infection cycle in Escherichia coli. Seven T4 proteins will be studied, UvsX, UvsY, UvsW, UvsW.1, Dda, gp32 and endonuclease VII. The recombination protein triad UvsX, UvsY and UvsW mediate the core of the homologous recombination reaction and are related to the eukaryotic proteins Rad51, Rad52 and Rad54, respectively. UvsW and Dda are helicases that translocate and/or unwind branched nucleic acid structures and have important roles in recombination and replication fork progression. Defects in helicases such as Bloom and Werner are known to cause cancer in humans, and there is evidence that UvsW and Dda may function very similarly to these molecules. UvsW.1 is a previously unknown T4 protein that we have identified, with a putative role in recombination. gp32 is the T4 single-stranded DNA binding protein that is known to have crucial roles in many aspects of T4 DNA metabolism. Finally, endonuclease VII resolves Holliday Junctions to complete the homologous recombination reaction. The mechanisms of, and interactions between, these seven proteins will be studied at the molecular level by a coordinated approach involving X-ray crystallography to study their structures, in vitro methods to study their individual functions and interactions, and in vivo methods to understand their biological roles. A considerable body of preliminary data has been obtained for this project that includes crystal and NMR structures, important preliminary crystals, purified proteins, demonstrations of biochemical activities, and in vivo function based on analysis of T4 mutants. PUBLIC HEALTH RELEVANCE: This project focuses on a fundamental DNA metabolic event that operates in all life forms, homologous recombination (HR). Traditionally associated with the propagation of genetic diversity, HR is now recognized as a major mechanism by which various forms of DNA damage are accurately and rapidly repaired. Many forms of cancer, notably breast cancer, are associated with defects in the HR machinery. The central events of HR are the pairing of DNA strands, the search for homology and the exchange of homologous DNA segments. Although the proteins that mediate this remarkable process are well characterized, the actual mechanism at the structural level is not well understood. The goals of the project are to study HR in the simple phage T4 system, to understand how the component T4 proteins coordinate the reaction, and to study how HR mediates DNA repair in T4 and in higher organisms. The project encompasses structural, genetics and biochemical techniques working in tandem to study these important biological questions.