Recombination and fork progression in bacteriophage T4

噬菌体 T4 的重组和分叉进展

• 批准号: 7267776
• 负责人: STEPHEN W WHITE
• 金额: $ 34.79万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7267776
• 关键词: 2' ,3' -Dideoxyadenosine; Bacteriophage T4; Binding; Biochemistry; Biological; Biological Assay; C-terminal; Cancer Etiology; Cells; Complex; Crystallization; DNA; DNA Binding; DNA Binding Domain; DNA Repair; DNA lesion; Data; Defect; Double Strand Break Repair; Ensure; Eubacterium; Eukaryota; Eukaryotic Cell; Genetic; Genetic Recombination; Genome Stability; Glutathione S-Transferase; Human; In Vitro; Individual; Laboratories; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Metabolism; Methodology; Methods; Middle Promoters; Molecular; Mutation; N-terminal; NMR Spectroscopy; Nucleic Acids; Open Reading Frames; Organism; Play; Predisposition; Prevention; Principal Investigator; Process; Protein Region; Proteins; Reaction; Resolution; Restart; Role; SF1; Site-Directed Mutagenesis; Specificity; Structure; Substrate Specificity; System; X-Ray Crystallography; base; design; experience; helicase; homologous recombination; human disease; in vivo; insight; interdisciplinary approach; interest; mutant; novel; nucleic acid structure; programs; repaired; research study; tool; transcription factor

DESCRIPTION (provided by applicant): The repair of DNA lesions such as double strand breaks (DSBs) is crucial to the stability of the genome, and we are proposing to study two processes that are fundamental to the prevention and repair of DSBs in all cells. The first is recombination-dependent replication (RDR) that is now recognized as a central mechanism in DNA metabolism that operates in many DNA repair scenarios. The second process is replication fork progression and the rescue of stalled forks. Stalled forks can lead to DSBs, and they need to be rapidly dealt with in the cell. We are interested in the underlying mechanisms that operate in these two processes, and will therefore study them in a very simple, well characterized organism, namely bacteriophage T4. Phage T4 is an ideal system for the multidisciplinary approach that we have designed, and there are many similarities between T4 and eukaryotic proteins. Defects in repair mechanisms lead to the accumulation of mutations that eventually result in cancer, and the proposed studies in T4 are therefore directly relevant to human disease. Five T4 proteins will be studied, UvsX, UvsY, UvsW, Dda and MotA. The recombinatorial proteins UvsX and UvsY mediate the T4 homologous recombination reaction that is required for RDR. UvsW and Dda are helicases that translocate and/or unwind branched nucleic acid structures and have important roles in RDR 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 be functionally homologous to these molecules. Finally, structural studies suggest that the T4 transcription factor MotA binds DNA in a novel fashion that is shared by UvsW. The mechanisms of these five proteins will be studied at the molecular level by a coordinated approach involving X-ray crystallography and NMR spectroscopy 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 have been obtained for this project that includes two high resolution structures, crystals, purified proteins and functional information from T4 mutants. The P.I. will direct the structural studies, and the co-P.I. will direct the in vivo studies. The in vitro analyses will be performed in both laboratories as appropriate.

描述（由申请人提供）：DNA病变（例如双链断裂（DSB））的修复对于基因组的稳定性至关重要，我们建议研究两个对于预防和修复所有细胞中DSB的基础的过程。 第一个是重组依赖性复制（RDR），该复制（RDR）现在被认为是在许多DNA修复方案中起作用的DNA代谢中的中心机制。 第二个过程是复制叉进程和停滞叉子的营救。 停滞的叉子可以导致DSB，并且需要在细胞中迅速处理。 我们对在这两个过程中运作的基本机制感兴趣，因此将以非常简单，表现良好的生物（即噬菌体T4）进行研究。噬菌体T4是我们设计的多学科方法的理想系统，T4和真核蛋白之间存在许多相似之处。 修复机制的缺陷导致突变的积累，最终导致癌症，因此T4中提出的研究与人类疾病直接相关。 将研究五种T4蛋白，UVSX，UVSY，UVSW，DDA和MOTA。 重组蛋白UVSX和UVSY介导了RDR所需的T4同源重组反应。 UVSW和DDA是旋转和/或不明智的分支核酸结构的解旋酶，在RDR和复制方向前进中具有重要作用。 众所周知，诸如Bloom和Werner之类的解旋酶缺陷会引起人类的癌症，并且有证据表明UVSW和DDA在功能上可能与这些分子同源。 最后，结构研究表明，T4转录因子MOTA以UVSW共享的新型方式结合DNA。 这五种蛋白质的机制将通过涉及X射线晶体学和NMR光谱的协调方法在分子水平上进行研究，以研究其结构，体外方法，以研究其个体功能和相互作用，以及体内方法以了解其生物学作用。 该项目获得了大量的初步数据，其中包括两个高分辨率结构，晶体，纯化的蛋白质和来自T4突变体的功能信息。 P.I.将指导结构研究和co-p.i。将指导体内研究。体外分析将在两个实验室中进行适当的作用。