Recombination and fork progression in bacteriophage T4

噬菌体 T4 的重组和分叉进展

• 批准号: 8097568
• 负责人: KENNETH N KREUZER
• 金额: $ 36.23万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097568
• 关键词: Affect; Bacteriophage T4; Biochemical; Biological; Biological Process; Biological Testing; Bypass; Cancer Etiology; Cleaved cell; Complement; Complex; Cruciform DNA; Crystallization; DNA; DNA Damage; DNA Repair; DNA-Binding Proteins; Data; Defect; Direct Repeats; Enzymes; Escherichia coli; Event; Filament; Gel; Genetic; Genetic Crosses; Genetic Recombination; Genetic Variation; Genome Stability; Goals; Human; Immigration; In Vitro; Individual; Infection; Investigation; Lead; Life; Malignant Neoplasms; Maps; Mediating; Mediator of activation protein; Membrane Proteins; Metabolic; Metabolism; Methods; Molecular; Mutagenesis; Mutation; Oligonucleotides; Organism; Pathology; Pathway interactions; Play; Predisposition; Process; Proteins; Reaction; Role; SF1; SS DNA BP; Sepharose; Site; Structure; Surface; System; Techniques; Testing; Triad Acrylic Resin; Work; X-Ray Crystallography; Yeasts; base; endodeoxyribonuclease VII; helicase; homologous recombination; human disease; in vitro Assay; in vivo; inhibitor/antagonist; interest; malignant breast neoplasm; mutant; nucleic acid structure; prevent; protein protein interaction; public health relevance; recombinase; repaired; research study

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.

描述（由申请人提供）： 同源重组是 DNA 代谢中的基本事件。长期以来，重组因其在产生遗传多样性方面的作用而受到认可，现在已知重组对于 DNA 修复和挽救停滞的复制叉至关重要。高等生物体中这些修复机制的缺陷会导致突变的积累，最终导致癌症，因此拟议的研究与人类疾病直接相关。我们有兴趣了解结构水平上重组的潜在机制，并建议在一种非常简单、特征明确的生物体（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，在大肠杆菌的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、gp32 和核酸内切酶 VII。重组蛋白三联体UvsX、UvsY和UvsW介导同源重组反应的核心，分别与真核蛋白Rad51、Rad52和Rad54相关。 UvsW 和 Dda 是易位和/或解旋分支核酸结构的解旋酶，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 的功能可能与这些分子非常相似。 UvsW.1 是我们已经鉴定出的一种以前未知的 T4 蛋白，假定在重组中发挥作用。 gp32 是 T4 单链 DNA 结合蛋白，已知在 T4 DNA 代谢的许多方面发挥着至关重要的作用。最后，核酸内切酶VII解析霍利迪连接点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方法进行研究，包括X射线晶体学研究其结构、体外方法研究其各自的功能和相互作用，以及体内方法了解其结构。生物学作用。该项目已获得大量初步数据，包括晶体和核磁共振结构、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。 公共健康相关性：该项目重点关注在所有生命形式中发挥作用的基本 DNA 代谢事件，即同源重组 (HR)。 HR 传统上与遗传多样性的传播相关，现在被认为是准确、快速修复各种形式的 DNA 损伤的主要机制。许多形式的癌症，尤其是乳腺癌，都与心率机制的缺陷有关。 HR 的中心事件是 DNA 链的配对、同源性的搜索以及同源 DNA 片段的交换。尽管介导这一非凡过程的蛋白质已得到很好的表征，但结构水平上的实际机制尚不清楚。该项目的目标是研究简单噬菌体 T4 系统中的 HR，了解 T4 蛋白成分如何协调反应，并研究 HR 如何介导 T4 和高等生物中的 DNA 修复。该项目涵盖结构、遗传学和生化技术，协同研究这些重要的生物学问题。