Study of the Catalytic Mechanism of T7 Gene 4 Helicase

描述（由申请人提供）：将检查DNA解旋酶解旋双链DNA的机制。DNA解旋酶介导DNA复制、修复、重组和转录，通过偶联核苷酸水解至单链DNA上的易位和双链DNA链的分离。在这里提出的研究中，由T7噬菌体编码的基因4蛋白将被用作模型解旋酶。T7基因4蛋白是复制型DNA解旋酶，其结合单链DNA作为六聚体，并且当其在5'至3'方向上易位时，使用从dTTP水解获得的能量解旋双链DNA。这项研究将集中在特定保守氨基酸的定点诱变和由此产生的遗传改变的蛋白质的生化分析。尽管包括T7基因4蛋白在内的DNA解旋酶已被生物化学表征，但对其催化机制和所涉及的氨基酸知之甚少。

DESCRIPTION (provided by applicant): The mechanism by which DNA helicases unwind double-stranded DNA will be examined. DNA helicases mediate DNA replication, repair, recombination, and transcription through the coupling of nucleotide hydrolysis to translocation on single-stranded DNA and separation of the strands of double-stranded DNA. In the research proposed here, the gene 4 protein encoded by T7 bacteriophage will be used as a model helicase. T7 gene 4 protein is a replicative DNA helicase that binds single-stranded DNA as a hexamer and, as it translocates in the 5' - to 3'- direction, unwinds double-stranded DNA using the energy obtained from the hydrolysis of dTTP. This research will focus on site-directed mutagenesis of specific conserved amino acids and biochemical analysis of the resulting genetically altered proteins. Although DNA helicases including T7 gene 4 protein have been characterized biochemically, there is little known about the catalytic mechanism and the amino acids involved.