Single Molecule Study of Helicase Mechanisms

描述(申请人提供)：解旋酶是将核苷酸结合和水解与核酸解离和易位相结合的分子马达。许多生物编码多种解旋酶，这些解旋酶对DNA复制、修复、重组、转录和翻译等基本细胞功能是必不可少的。几种人类遗传疾病也与DNA解旋酶的突变有关，来自人类病原体的解旋酶正被积极用作药物靶标。我们建议使用我们和其他人开发的强大的单分子荧光技术来研究两类解旋酶的分子机制。我们的方法已经产生了几个惊喜，并提供了以前无法获得的复杂生化过程的数据。例如，利用单分子荧光共振能量转移，我们发现当大肠杆菌Rep解旋酶单体在其3‘-5’易位过程中遇到阻碍时，它会迅速回到起始位置，并多次重复相同的循环，称为“重复穿梭”。进一步的单分子分析表明了重复穿梭的合理的物理机制和生物学意义(Myong等人，《自然》，2005年)。对于超家族1解旋酶(Rep/UvrD/PcrA)，我们的目标是(1)揭示单链DNA易位和重复穿梭的机制，(2)确定观察到的大规模解旋酶构象变化的功能和调节作用，(3)可视化解旋过程中解旋酶单体之间的配位并直接测量步长。(4)我们还将研究人类丙型肝炎病毒超家族2解旋酶NS3的两级阶跃机制，并深入了解其重复解离和逐步再退火等新行为。(5)我们还将研究解旋酶如何与其他蛋白质一起发挥作用。(6)最后，我们将进一步发展我们的单分子技术，用于多色成像和与微流控设备的集成。在我们的所有研究中，体相生化实验、结晶学和通过合作进行的计算研究将帮助我们设计和解释我们的测量结果。

DESCRIPTION (provided by applicant): Helicases are molecular motors that couple nucleotide binding and hydrolysis to nucleic acid unwinding and translocation. Many organisms encode multiple helicases that are essential to fundamental cellular functions such as DNA replication, repair, recombination, transcription and translation. Several human genetic disorders have also been linked to mutations in DNA helicases and helicases from human pathogen are being actively pursued as drug target. We propose to study the molecular mechanisms of two classes of helicases using the powerful single molecule fluorescence techniques that we and others have developed. Our approaches have already yielded several surprises and provided previously unattainable data on complex biochemical processes. For example, using single molecule fluorescence resonance energy transfer, we discovered that when a monomer of E. coli Rep helicases encounters a blockade during its 3'-5' translocation, it snaps back to the beginning site and repeats the same cycle multiple times, termed 'repetitive shuttling'. Further single molecule analysis suggested a plausible physical mechanism and biological implications of repetitive shuttling (Myong et al, Nature, 2005). On superfamily 1 helicases (Rep/UvrD/PcrA), we aim to (1) uncover the mechanisms of single stranded DNA translocation and repetitive shuttling, (2) determine the functional and regulatory roles of the observed large scale helicase conformation changes, and (3) visualize the coordination between helicase monomers during unwinding and measure the step sizes directly. (4) We will also study the mechanisms of two-tiered stepping of NS3, a superfamily 2 helicase from human pathogen Hepatitis C virus, and gain insight into the novel behaviors such as repetitive unwinding and gradual re-annealing. (5) We will also study how helicases function together with other proteins. (6) Finally, we will further develop our single molecule techniques for multicolor imaging and integration with microfluic devices. In all of our studies, bulk-phase biochemical experiments, crystallography, and computational studies through collaboration will help us design and interpret our measurements.