Study of the Catalytic Mechanism of T7 Gene 4 Helicase

描述(由申请人提供)：将检查DNA解旋酶解开双链DNA的机制。DNA解旋酶通过核苷酸水解与单链DNA易位和双链DNA链分离的偶联来介导DNA的复制、修复、重组和转录。在本文提出的研究中，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蛋白是一种复制DNA解旋酶，它以六聚体的形式与单链DNA结合，当它向5‘-3’方向移位时，利用dTTP水解获得的能量解开双链DNA。这项研究将集中在特定保守氨基酸的定点突变和由此产生的基因改变蛋白质的生化分析上。尽管包括T7基因4蛋白在内的DNA解旋酶已经被生化鉴定，但对其催化机制和涉及的氨基酸却知之甚少。

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.