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代谢中的一个基本事件。长期以来，重组以其在产生遗传多样性中的作用而认可，现在已知重组对于DNA修复至关重要，并营救了停滞的复制叉。这些修复机制的缺陷导致突变的积累，最终导致癌症，因此所提出的研究与人类疾病直接相关。我们有兴趣了解结构水平上的重组的潜在机制，并建议在非常简单，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，以在大肠杆菌中的感染周期中产生所需的DNA水平。 将研究七个T4蛋白，UVSX，UVSY，UVSW，UVSW.1，DDA，GP32和核酸内切酶VII。重组蛋白三合会UVSX，UVSY和UVSW分别介导同源重组反应的核心，并分别与真核蛋白Rad51，Rad52和Rad54有关。 UVSW和DDA是旋转和/或不明智的分支核酸结构的解旋酶，并且在重组和复制方叉进程中具有重要作用。众所周知，诸如Bloom和Werner等解旋酶的缺陷在人类中会引起癌症，并且有证据表明UVSW和DDA可能与这些分子非常相似。 UVSW.1是我们已经确定的先前未知的T4蛋白，在重组中起着推定的作用。 GP32是T4单链DNA结合蛋白，已知在T4 DNA代谢的许多方面具有至关重要的作用。最后，核酸内切酶VII解决了Holliday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通过涉及X射线晶体学研究其结构的协调方法在分子水平上进行研究，以研究其个体功能和相互作用的体外方法，以及在体内方法，以了解其生物学作用。该项目已经获得了包括晶体和NMR结构，重要的初步晶体，纯化蛋白质，生化活性的演示以及基于T4突变体分析的体内功能的大量初步数据。 公共卫生相关性：该项目着重于以所有生命形式，同源重组（HR）运作的基本DNA代谢事件。传统上，与遗传多样性的传播相关，人力资源现在被公认为是一种主要机制，通过这种机制，通过这种机制进行了各种形式的DNA损伤，可以准确迅速修复。许多形式的癌症，尤其是乳腺癌，与人力资源机械中的缺陷有关。 人力资源的中心事件是DNA链的配对，寻找同源性和同源DNA片段的交换。尽管介导这种非凡过程的蛋白质的表征很好，但在结构层面上的实际机制尚不清楚。该项目的目标是在简单的噬菌体T4系统中研究HR，以了解组分T4蛋白如何协调反应，并研究HR如何介导T4和更高生物体中的DNA修复。该项目涵盖了与研究这些重要生物学问题一起研究的结构，遗传学和生化技术。