KINETIC ANALYSIS OF THE HELICASE/NTPASE

描述：DNA解旋酶是一种普遍存在的蛋白质，是 将双链DNA解离成单链DNA的活体过程 能量耦合到NTP的水解。DNA解旋酶在所有 DNA新陈代谢过程和DNA解旋酶缺陷已被发现 对人类疾病负责，如色素性干皮病、Cockayne氏症 综合征和布卢姆综合征。这项研究的长期目标是 了解这种能量转导酶在动力学上的作用机制， 热力学层面和结构层面。调查人员已经选择了 以噬菌体T7 DNA解旋酶为模型系统进行研究。T7DNA解旋酶 是T7噬菌体参与DNA复制的主要解旋酶。它 是由相同亚基组成的环状六角体，中心有一个孔 通过它与单链DNA结合。每个六聚体只结合3个NTP，并且 研究人员的平衡DNA结合研究表明， 在NTP状态下，解旋酶与DNA的相互作用更紧密 “NDP--国家”。单链DNA不对称地结合到与之相互作用的六聚体上 在任何给定的时间只有一个或两个亚基。调查人员提出， NTP被解旋酶六聚体以协调的方式水解，从而导致 到DNA解旋酶转位所需的“DNA结合-释放”。 在这个模型中，每个单体或二聚体与DNA按顺序相互作用。 催化DNA解离所需的易位的方式。NTPase 反应为DNA结合-释放过程提供了“开关”。 建议进行实验，以测试和区分各种机制 通过测定NTP与DNA结合的单周转动力学和 NTPase反应的稳态前动力学。众所周知，GP4 解旋酶需要在双链的一端有两个非互补的单链DNA尾巴 DNA(叉状DNA)以启动DNA解开。理解的第一步 解旋机制是确定解旋酶与 叉子DNA。研究人员提议进行实验，以研究 DNA解链中的3‘-尾，并决定DNA解链的速度。 建议进行的研究有以下具体目的：i)衡量 停流法与核苷酸和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-流动，以了解dTTP结合中的配位，其水解， 和产物通过六聚体的亚基解离，以及iii)到 研究3‘-单链DNA尾巴的作用，并测量内在率 DNA解开。

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.