Recombination and fork progression in bacteriophage T4

噬菌体 T4 的重组和分叉进展

• 批准号: 6931049
• 负责人: STEPHEN W WHITE
• 金额: $ 34.62万
• 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/6931049
• 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(申请人提供)：DNA损伤的修复，如双链断裂(DSB)对基因组的稳定性至关重要，我们建议研究两个过程，这两个过程是预防和修复所有细胞中DSB的基础。第一种是重组依赖复制(RDR)，它现在被认为是DNA新陈代谢的核心机制，在许多DNA修复场景中运行。第二个过程是复制叉子进程和抢救停滞的叉子。卡住的叉子可能会导致DSB，它们需要在单元中快速处理。我们对这两个过程中的潜在机制很感兴趣，因此我们将在一个非常简单、特性良好的有机体中研究它们，即噬菌体T4。噬菌体T4是我们设计的多学科方法的理想系统，而且T4与真核蛋白有许多相似之处。修复机制的缺陷会导致突变的积累，最终导致癌症，因此T4中拟议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RDR所需的T4同源重组反应。UvsW和Dda是转位和/或解开支链核酸结构的解旋酶，在RDR和复制分叉过程中发挥重要作用。解旋酶的缺陷，如Bloom和Werner，已知会导致人类癌症，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以一种新的方式与DNA结合，这与UvsW相同。这五种蛋白质的作用机制将通过X射线结晶学和核磁共振光谱学相结合的方法在分子水平上进行研究，包括研究它们的结构，体外方法研究它们的单独功能和相互作用，以及体内方法了解它们的生物学作用。这个项目已经获得了相当多的初步数据，包括两个高分辨结构、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，而co-P.I.将指导体内研究。体外分析将酌情在两个实验室进行。