Recombination and fork progression in bacteriophage T4

噬菌体 T4 的重组和分叉进展

• 批准号: 6782336
• 负责人: STEPHEN W WHITE
• 金额: $ 34.88万
• 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 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6782336
• 关键词: DNA repair; DNA replication origin; X ray crystallography; bacterial proteins; bacteriophage T4; genetic recombination; mass spectrometry; nuclear magnetic resonance spectroscopy; nucleic acid metabolism; protein structure function; site directed mutagenesis

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）：双链断裂 (DSB) 等 DNA 损伤的修复对于基因组的稳定性至关重要，我们建议研究对于所有细胞中预防和修复 DSB 至关重要的两个过程。 第一个是重组依赖性复制 (RDR)，它现在被认为是 DNA 代谢的核心机制，在许多 DNA 修复场景中发挥作用。 第二个过程是复制叉进展和停滞叉的救援。 停滞的分叉可能会导致 DSB，需要在单元中快速处理。 我们对这两个过程中运作的基本机制感兴趣，因此将在一个非常简单、特征明确的生物体（即噬菌体 T4）中研究它们。噬菌体 T4 是我们设计的多学科方法的理想系统，T4 和真核蛋白之间有许多相似之处。 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 T4 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 RDR 所需的 T4 同源重组反应。 UvsW 和 Dda 是易位和/或解旋分支核酸结构的解旋酶，在 RDR 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 可能在功能上与这些分子同源。 最后，结构研究表明 T4 转录因子 MotA 以 UvsW 所共有的一种新颖方式结合 DNA。 这五种蛋白质的机制将通过协调方法在分子水平上进行研究，其中包括 X 射线晶体学和核磁共振波谱法来研究它们的结构，体外方法来研究它们各自的功能和相互作用，以及体内方法来了解它们的生物学作用。 该项目已获得大量初步数据，包括来自 T4 突变体的两种高分辨率结构、晶体、纯化蛋白质和功能信息。 P.I.将指导结构研究，共同 P.I.将指导体内研究。体外分析将酌情在两个实验室进行。