Single Molecule Studies of DNA Helicases

描述(申请人提供)：这项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。为了实现这一目标，该提案被细分为三个主要问题。结构不同的螺旋酶通过什么机制促进复杂DNA底物的加工？各种解旋酶基序是如何实现转位和链分离的？DNA马达在转移和解开DNA时所施加的力是什么？对于大多数实验，单分子技术将被用来回答这三个问题。为了给前两个问题提供答案，比安科博士将结合光学镊子和荧光显微镜，直接观察这些运动中的动态纳米机器。为解决第三个问题而设计的实验将利用原子力显微镜来测量DNA解旋酶在移位和DNA解离过程中的作用力。比安科博士将使用这些单分子技术来研究四种DNA马达蛋白质，这些蛋白质被选中，以提供关于这些酶运动动力学的细节和细微差别的独特和补充信息。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。预计对这些纳米机器的实时直接观察将为DNA解旋酶的生化机制提供新的见解，DNA解旋酶是一类对DNA新陈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导致DNA代谢过程异常的分子事件的总体认识。有证据表明，导致Bloom综合征、Cockayne综合征、Werner综合征和着色性干皮病的基因缺陷都被鉴定为DNA解旋酶突变，从而强调了研究DNA解旋酶的重要性。

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.