HELICASE CATALYZED DNA UNWINDING

描述：(摘自申请者摘要)DNA解旋酶是一种依赖于ATP的马达 解开双链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。首席调查员和他的小组建议继续研究 在两个大肠杆菌DNA解旋酶中，Rep和UvrD(解旋酶II)，两者都出现 为了发挥同源二聚体的作用并在复制和修复中发挥作用， 分别进行了分析。总体目标是从分子水平上了解 解旋酶解离双链dna和转位的机制(S) 以及这些过程是如何与ATP结合和水解相耦合的。 将使用定量生化和生物物理方法来检查 在功能上重要的相互作用的平衡和动力学 DNA解离，如DNA和核苷酸结合、ATP水解酶和蛋白质 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 多亚单位酶。首席调查员和他的团队提出了一项 Rep二聚体如何移位和解开双链的“亚单位切换”模型 作出了许多可验证的预测的DNA；许多拟议的研究 专注于测试这一模型和其他模型。 我们将使用瞬时动力学方法(停流荧光 和化学淬火流动)来检查预稳态动力学 以及Rep和UvrD二聚体与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 放松反应。DNA结合的热力学、动力学和机理 也将进行研究。同时，他们将检测Rep和UvrD催化的 解开合成DNA底物，目标是开发完整的 解开合成DNA底物的动力学机制，目标是 开发一种用于展开的全动力机构。他们最新的x射线晶体 Rep-ssDNA复合体的结构(与G.Waksman合作)具有 提供了重要的结构洞察力，将有助于Rep和UrvD的设计 突变体以测试蛋白质不同结构域的功能重要性 用于DNA和ATP的结合、ATP的水解、二聚化和DNA解离。

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.