Single Molecule Studies of DNA Helicases

描述（由申请人提供）：本研究提案的目标是在单分子水平上深入了解DNA解旋酶的生化机制。为实现这一目标，本提案分为三个主要问题。结构上不同的解旋酶通过什么机制促进复杂DNA底物的加工？不同的解旋酶基序如何实现移位和链分离？当DNA马达进行DNA移位和解旋时，它们施加了什么力？对于大多数实验，单分子技术将用于回答这三个问题。为了回答前两个问题，Bianco博士将结合联合收割机和荧光显微镜，直接观察这些动态纳米机器的运动。旨在解决问题三的实验将利用原子力显微镜来测量DNA解旋酶在易位和DNA解旋过程中的操作力。Bianco博士将使用这些单分子技术来研究四种DNA马达蛋白，这些蛋白被选择来提供关于这些酶运动动力学的细节和细微差别的独特和互补信息。这些大肠杆菌酶是DNA解旋酶RecBCD、RecG、RuvAB和I型限制酶EcoR124I。预计在真实的时间内直接观察这些纳米机器将为DNA解旋酶的生化机制提供新的见解，DNA解旋酶是一类对DNA代谢至关重要的核酸马达。此外，对这些蛋白质的更详细的了解将有助于对异常DNA代谢过程的分子事件的一般理解。研究DNA解旋酶的重要性是强调的证据表明，遗传缺陷导致布卢姆氏综合征，科凯恩氏综合征，沃纳综合征，着色性干皮病，都被确定为DNA解旋酶的突变。

DESCRIPTION (provided by applicant): The goal of this research proposal is to provide insight into the biochemical mechanisms of DNA helicases at the single molecule level. To achieve this goal, this proposal is subdivided into three primary questions. By what mechanism do structurally distinct helicases facilitate processing of complex DNA substrates? How do the various helicase motifs achieve translocation and strand separation? What are the forces exerted by DNA motors as they proceed to translocate and unwind DNA? For the majority of experiments, single molecule techniques will be used to answer these three questions. To provide answers to the first two questions Dr. Bianco will combine optical tweezers and fluorescence microscopy to directly visualize these dynamic nanomachines in motion. The experiments designed to address question three will take advantage of atomic force microscopy to measure the forces in operation for DNA helicases during translocation and DNA unwinding. Dr. Bianco will use these single molecule techniques to study four DNA motor proteins that have been selected to provide both unique and complementary information on the details and nuances of the dynamics of motion of these enzymes. These Escherichia coli enzymes are the DNA helicases RecBCD, RecG, RuvAB and the type I restriction enzyme EcoR124I. It is anticipated that direct observation of these nanomachines in real time will provide novel insights into the biochemical mechanism of DNA helicases, a class of nucleic acid motors that are of fundamental importance to DNA metabolism. In addition, a more detailed understanding of these proteins will contribute to a general appreciation of the molecular events responsible for aberrant DNA metabolic processes. The importance of studying DNA helicases is emphasized by evidence demonstrating that the genetic defects leading to Bloom's syndrome, Cockayne's syndrome, Werner syndrome, and Xeroderma pigmentosum, have all been identified as mutations in DNA helicases.