HELICASE CATALYZED DNA UNWINDING

描述：（来自申请人的摘要）DNA解旋酶是ATP依赖性马达 解旋双链DNA形成单链DNA的蛋白质 复制，重组和修复所需的中间体， 有机体首席研究员和他的小组建议继续研究 两个E。大肠杆菌DNA解旋酶，Rep和UvrD（解旋酶II），这两个酶都出现在 作为同源二聚体并在复制和修复中起作用， 分别总的目标是获得分子的理解， 这些DNA解旋酶解开双链体DNA并易位的机制 沿着DNA以及这些过程如何与ATP结合和水解偶联。 定量生物化学和生物物理方法将用于检查 平衡和动力学的相互作用，是功能上重要的， DNA解旋，如DNA与核苷酸结合、ATP水解和蛋白质 自我组装这需要研究人员了解分子细节 已知的变构相互作用是这些功能的关键 多亚基酶首席研究员和他的团队提出了一个 Rep二聚体如何易位和解旋双链体的“亚基转换”模型 DNA，使一些可测试的预测;许多建议的研究 专注于测试这个模型和其他模型。 我们将使用瞬态动力学方法（停流荧光 和化学猝灭流）来检查预稳态动力学 以及Rep和UvrD二聚体对ATP结合和水解的机制。 各种DNA连接状态，其中一些被认为是DNA中的中间体 解旋反应DNA结合的热力学、动力学和机理 也将被研究。同时，他们将研究Rep和UvrD催化的 解旋合成的DNA底物，目的是开发一个完整的 合成DNA底物解旋动力学机制 发展出一个完整的解绕动力学机制。他们最近的x射线晶体 Rep-ssDNA复合物的结构（与G. Waksman） 提供了重要的结构见解，并将有助于设计Rep和UrvD 突变体来测试蛋白质的不同结构域的功能重要性 用于DNA和ATP结合、ATP水解、二聚化和DNA解旋。

DESCRIPTION: (From applicant's abstract)DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for replication, recombination and repair in all organisms. The principal investigator and his group propose to continue studies of two E. coli DNA helicases, Rep and UvrD (Helicase II), both of which appear to function as homo-dimers and function in replication and repair, respectively. The overall goal is to obtain a molecular understanding of the mechanism(s) by which these DNA helicases unwind duplex DNA and translocate along DNA and how these processes are coupled to ATP binding and hydrolysis. Quantitative biochemical and biophysical approaches will be used to examine the equilibria and kinetics of the interactions that are functionally important for DNA unwinding, such as DNA and nucleotide binding, ATP hydrolysis and protein self-assembly. This requires investigators to understand the molecular details of the known allosteric interactions that are key to the function of these multisubunit enzymes. The principal investigator and his group have proposed a "subunit switching" model for how the Rep dimer translocates and unwinds duplex DNA that makes a number of testable predictions; many of the proposed studies are focused on testing this and other models. We will use transient kinetic approaches (stopped-flow fluorescence and chemical quenched-flow) to examine the pre-steady state kinetics and mechanism of ATP binding and hydrolysis by Rep and UvrD dimers in various DNA ligation states, some of which are proposed intermediates in DNA unwinding reactions. The thermodynamics, kinetics and mechanism of DNA binding will also be studied. In parallel, they will examine Rep and UvrD catalyzed unwinding of synthetic DNA substrates with the goal of developing a full kinetic mechanism for unwinding of synthetic DNA substrates with goal of developing a full kinetic mechanism for unwinding. Their recent x-ray crystal structure of Rep-ssDNA complexes (in collaboration with G. Waksman) has provided important structural insight and will aid the design of Rep and UrvD mutants to test the functional importance of different domains of the proteins for DNA and ATP binding, ATP hydrolysis, dimerization and DNA unwinding.