Single Molecule Studies of DNA Helicases

这项研究的目的是提供深入了解DNA解旋酶的生化机制 在单分子水平上。 为实现这一目标，本提案分为三个主要问题。通过什么机制 结构上不同的解旋酶促进复杂DNA底物的加工？各种解旋酶如何 基序实现易位和链分离？DNA马达在运动时所施加的力是什么？ 转移并解开DNA 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，我们将联合收割机和荧光显微镜相结合， 直接将这些动态纳米机器可视化。针对问题三的实验 将利用原子力显微镜来测量DNA解旋酶在操作过程中的力， 易位和DNA解旋。我们将利用这些单分子技术来研究四种DNA马达 这些蛋白质被选择来提供关于细节和细微差别的独特和互补信息， 这些酶的运动动力学。这些大肠杆菌酶是DNA解旋酶RecBCD， RecG、RuvAB和I型限制性内切酶EcoR124I。 预计这些纳米机器在真实的时间的直接观察将提供新的见解， DNA解旋酶的生化机制，一类具有根本重要性的核酸马达 to DNA DNA metabolism新陈代谢.此外，对这些蛋白质的更详细的了解将有助于对这些蛋白质进行全面的研究。 负责异常DNA代谢过程的分子事件的评价。 研究DNA解旋酶的重要性是强调证据表明，遗传 导致Bloom综合征、Cockayne综合征、Werner综合征和干皮病的缺陷 色素瘤，都被确定为DNA解旋酶的突变。

The goal of this research proposal is to provide insight into the biochemical mechanisms ofDNA 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, we 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. We 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.