KINETIC ANALYSIS OF THE HELICASE-NTPASE

描述：DNA解旋酶是普遍存在的蛋白质， 将双链DNA解旋成单链DNA的方法， 能量耦合到NTP水解。 DNA解旋酶在所有 已经发现DNA代谢过程和DNA解旋酶的缺陷 导致人类疾病，如着色性干皮病， Bloom综合征。 这项研究的长期目标是 了解这种能量转换酶的动力学机制， 热力学和结构水平。 调查人员选择了 噬菌体T7 DNA解旋酶作为模型系统进行研究。 T7 DNA解旋酶 是参与DNA复制的T7噬菌体的主要解旋酶。 它 是相同亚基的环状六聚体， 通过它结合ssDNA。 每个六聚体仅结合3个NTPs， 研究人员的平衡DNA结合研究表明， 解旋酶在“NTP状态”下与DNA的相互作用比在“NTP状态”下更紧密。 “NDP州”。 ssDNA不对称地与六聚体结合， 在任何给定时间只有一个或两个子单元。 调查人员提出， NTP被解旋酶六聚体以协调的方式水解， 到解旋酶在DNA上移位所需的“DNA结合-释放”。 在这个模型中，每个单体或二聚体与DNA以连续的方式相互作用。 以催化DNA解旋所需的易位的方式。 新台币 反应为DNA结合-释放过程提供了“开关”。 提出实验来测试和区分各种机制 通过测量NTP和DNA结合的单周转动力学， NTT反应的稳态前动力学。 据了解，GP 4 解旋酶在双链体的一端需要两个非互补的ssDNA尾 DNA（fork DNA）启动DNA解旋。 理解的第一步 解旋机制是决定解旋酶与 叉子的DNA 研究人员提出实验来研究 DNA解旋中的3 ′-尾，并测定DNA解旋的速率。 建议进行的研究有以下具体目标： 通过停流法测定核苷酸和DNA结合的动力学，ii） 用快速化学法测定dTTP水解的稳态前动力学 猝灭流，以了解dTTP结合，其水解， 和通过六聚体的亚基的产物解离，和iii） 研究3 '-ssDNA尾的作用，并测量 DNA解旋。

DESCRIPTION: DNA helicases are ubiquitous proteins that are required in vivo for unwinding duplex DNA into single-stranded DNAs, a process energetically coupled to NTP hydrolysis. DNA helicases are important in all processes of DNA metabolism and a defect in DNA helicase has been found to be responsible for human diseases such as xeroderma pigmentosa, cockayne's syndrome, and bloom syndrome. The long term goal of the research is to understand the mechanism of this energy transducing enzyme at the kinetic, thermodynamic, and structural level. The investigators have chosen bacteriophage T7 DNA helicase to study as a model system. T7 DNA helicase is the primary helicase of T7 bacteriophage involved in DNA replication. It is a ring-shaped hexamer of identical subunits that has a central hole through which it binds ssDNA. Each hexamer binds only 3 NTPs, and the equilibrium DNA binding studies by the investigator have shown that the helicase interacts with the DNA more tightly in the "NTP-state" vs. the "NDP-state". The ssDNA binds asymmetrically to the hexamer interacting with only one or two subunits at any given time. The investigators propose that NTP is hydrolyzed by the helicase hexamer in a coordinated manner that leads to "DNA bind-release" required for translocation of the helicase on the DNA. In this model, each monomer or dimer interacts with the DNA in a sequential manner to catalyze translocation required for DNA unwinding. The NTPase reaction provides the "switch" for the DNA bind-release process. Experiments are proposed to test and distinguish between various mechanisms by measuring the single-turnover kinetics of NTP and DNA binding and the presteady state kinetics of the NTPase reaction. It is known that gp4 helicase requires two noncomplementary ssDNA tails at one end of the duplex DNA (fork DNA) to initiate DNA unwinding. The first step to understanding the unwinding mechanism is to determine the interaction of the helicase with the fork DNA. The investigators propose experiments to study the role of the 3'-tail in DNA unwinding, and determine the rate of DNA unwinding. Studies are proposed with the following specific aims: i) to measure the kinetics of nucleotide and DNA binding by stopped-flow methods, ii) to measure the presteady state kinetics of dTTP hydrolysis by rapid chemical quench-flow to understand the coordination in dTTP binding, its hydrolysis, and product dissociation by the subunits of the hexamer, and iii) to investigate the role of the 3'-ssDNA tail, and measure the intrinsic rate of DNA unwinding.