Helicase Catalyzed DNA Unwinding

描述（由申请人提供）： DNA解旋酶是ATP依赖的马达蛋白，其解旋双链DNA以形成所有生物体中DNA代谢所需的单链（ss）DNA中间体。我们研究了大肠杆菌三种非六聚体SF 1 DNA解旋酶的DNA解旋和DNA转位的动力学机制。coil、Rep、UvrD和RecBCD，它们分别在复制、修复和重组中起作用。RecBCD是含有两个SF 1解旋酶（B和D）的异源三聚体复合物。总体目标是获得对这些DNA解旋酶解链双链体DNA、沿沿着移位以及这些过程如何与ATP结合和水解偶联的动力学机制的分子理解。我们的预稳态动力学研究表明，Rep和UvrD解旋酶作为二聚体的功能，即使这些酶的单体可以有效地沿着ss-DNA易位，因此ss-DNA易位单体和DNA解旋二聚体之间的关系将被调查。我们将使用瞬态动力学方法（停流荧光和化学猝灭流），以检查前稳态动力学和机制的ss-DNA易位，ATP结合和水解的Rep和UvrD单体在ss-DNA易位，并比较这些功能解旋酶二聚体的DNA解旋的动力学机制，与发展一个完整的解旋动力学机制的目标。DNA结合，ATP水解和DNA解旋的RecBCD和RecBC解旋酶也将进行机械检查。将进行热力学研究，以了解这些解旋酶与DNA结合的能量学，以及DNA结合能如何用于DNA解旋。我们还将研究这些酶的突变体，包括Rep的组成型二聚体，以及MutL通过UvrD刺激DNA解旋的机制。这些合奏研究将补充单分子研究的DNA结合和解旋和结构研究的X射线晶体学。

DESCRIPTION (provided by applicant): DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss)DNA intermediates required for DNA metabolism in all organisms. We are studying the kinetic mechanisms of DNA unwinding and DNA translocation of three non-hexameric SF1 DNA helicases from E. coil, Rep,UvrD, and RecBCD, which function in replication, repair, and recombination, respectively. RecBCD is a hetero-trimeric complex containing two SF1 helicases (B and D). The overall goal is to obtain a molecular understanding of the kinetic mechanism(s) by which these DNA helicases unwind duplex DNA, translocate along DNA and how these processes are coupled to ATP binding and hydrolysis. Our pre-steady state kinetic studies indicate that Rep and UvrD helicases function as dimers, even though monomers of these enzymes can translocate efficiently along ss-DNA, hence the relationship between ss-DNA translocation by monomers and DNA unwinding by dimers will be investigated. We will use transient kinetic approaches(stopped-flow fluorescence and chemical quenched-flow) to examine the pre-steady state kinetics and mechanism of ss-DNA translocation, ATP binding and hydrolysis by Rep and UvrD monomers during ss-DNA translocation, and compare these to the kinetic mechanism for DNA unwinding by the functional helicase dimers, with the goal of developing a full kinetic mechanism for unwinding. DNA binding, ATP hydrolysis and DNA unwinding by RecBCD and RecBC helicases will also be examined mechanistically. Thermodynamic studies will be performed to understand the energetics of the binding of these helicases to DNA, and how DNA binding energy is used in DNA unwinding. We will also examine mutants of these enzymes, including constitutive dimers of Rep, and the mechanism by which MutL stimulates DNA unwinding by UvrD. These ensemble studies will be complemented by single molecule studies of DNA binding and unwinding and structural studies by x-ray crystallography.