Study of the Catalytic Mechanism of T7 Gene 4 Helicase

描述（由申请人提供）：DNA解旋酶解开双链DNA的机制将被检查。DNA解旋酶通过核苷酸水解与单链DNA易位和双链DNA链分离的耦合作用介导DNA的复制、修复、重组和转录。本研究拟采用T7噬菌体编码的基因4蛋白作为解旋酶模型。T7基因4蛋白是一种复制性DNA解旋酶，它结合单链DNA作为六聚体，当它在5‘到3’方向上易位时，利用dTTP水解获得的能量解开双链DNA。本研究将集中于特定保守氨基酸的定点诱变和由此产生的基因改变蛋白的生化分析。虽然包括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.