Single Molecule Study of Helicase Mechanisms

描述（由申请人提供）：解旋酶是将核苷酸结合和水解与核酸解旋和易位偶联的分子马达。许多生物体编码多种解旋酶，这些解旋酶对于基本细胞功能如DNA复制、修复、重组、转录和翻译是必需的。几种人类遗传疾病也与DNA解旋酶的突变有关，并且来自人类病原体的解旋酶正被积极地用作药物靶标。我们建议使用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的生物化学过程的数据。例如，使用单分子荧光共振能量转移（FRET），我们发现PcrA解旋酶以单碱基步骤卷绕DNA，形成DNA环，并且同时有效地去除与DNA结合的其他蛋白质（Park等人，Cell，2010）。我们还发现，来自人类病原体丙型肝炎病毒的NS 3解旋酶使用弹簧加载机制解旋DNA（Myong等人，Science，2007）。超家族1解旋酶（Rep/UvrD/PcrA），我们的目标是（1）揭示ATP水解和DNA易位之间的化学-机械耦合的机制和功能重要性，（2）使用结合单荧光团检测的高分辨率光镊的混合仪器关联DNA解旋和酶构象变化，（3）通过基于成像的定位探测解旋酶和其它DNA结合蛋白质之间相遇的结果。在复制型六聚体解旋酶方面，我们的目标是（1）确定T7 gp 4、DnaB样解旋酶G40 P和SV 40大T抗原（LTag）的DNA解旋和亚基间协调的步长，（2）研究解旋酶滑动的机制以及其他相互作用蛋白如何调节滑动事件，（3）检测环状解旋酶在沿着双链DNA移位过程中是否绕DNA轴旋转，和（4）使用四色单分子FRET探测从起始序列以相反方向解旋的LTag的两个六聚体的潜在配位。在我们所有的研究中，通过合作获得的体相生化实验和结构数据将帮助我们设计和解释我们的测量。

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. 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 (FRET), we discovered that PcrA helicase reels in DNA in single base steps, forming a DNA loop, and at the same time efficiently removing other proteins bound to the DNA (Park et al, Cell, 2010). We also discovered that NS3 helicase from a human pathogen, hepatitis C virus, unwinds DNA using a spring-loaded mechanism (Myong et al, Science, 2007). On superfamily 1 helicases (Rep/UvrD/PcrA), we aim to (1) uncover the mechanism and functional importance of chemo-mechanical coupling between ATP hydrolysis and DNA translocation, (2) correlate DNA unwinding and enzyme conformational changes using a hybrid instrument combining ultrahigh resolution optical tweezers with single fluorophore detection, (3) probe consequences of an encounter between a helicase and other DNA bound proteins via imaging-based localization. On replicative hexameric helicases, we aim to (1) determine the step size of DNA unwinding and inter-subunit coordination of T7 gp4, DnaB-like helicase G40P and SV40 Large T antigen (LTag), (2) investigate the mechanism of helicase slipping and how other interacting proteins modulate the slipping events, (3) test if the ring-shaped helicase rotates around the DNA axis during its translocation along double stranded DNA, and (4) probe for a potential coordination two hexamers of LTag unwinding from the origin sequence in opposite directions using four-color single molecule FRET. In all of our studies, bulk-phase biochemical experiments and structural data through collaboration will help us design and interpret our measurements.