Single Molecule Study of Helicase Mechanisms

我们将使用新的荧光分析法研究DNA解旋酶的构象变化和功能。 该测定包括单分子荧光共振能量转移（smFRET）、淬灭、单分子计数和系综FRET。单分子技术可以在真实的时间内测量解旋酶活性，而不需要系综平均。 这些技术也适用于许多生物系统，事实上，我们最近已经使用它们来检测RNA分子的构象变化，并研究核酶的折叠途径和催化作用。 特异性解旋酶将来自包括大肠杆菌Rep和UvrD解旋酶的SF 1解旋酶家族。 所获得的机械信息也将有助于其他解旋酶的研究。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。 解旋酶也与其他分子马达共享许多特性。 因此，解旋酶的基本理解是科学和医学的重要性。 在以前的研究中，我们已经使用了smFRET染料连接到DNA研究E。coli Rep解旋酶。 我们将DNA固定在聚合物涂层的表面上，这使得观察时间延长，同时保持几乎完整的生化活性。 解旋只有几个碱基对，因此连接到DNA的两个染料之间的距离变化可以通过smFRET检测。 我们还发现了一些新的构象，并确定了它们之间的波动率。 使用这些技术的进一步工作，在这里提出，准备回答许多基本问题。(1)解旋酶的寡聚化是DNA解旋所必需的吗？如果是，为什么？(2)每个生化循环有多少碱基对解旋，什么因素影响解旋的持续性？ ATP水解与解旋的耦合有多紧密？(3)解旋酶构象变化的功能作用是什么？核苷酸和DNA的结合如何影响它们？(4)DNA解旋方向性的起源是什么？为了实现这些目标，我们将使用系综和单分子测量的染料连接到DNA和解旋酶的各个网站。 具体地，将在DNA上的位点之间、解旋酶和DNA之间、解旋酶和ATP之间、解旋酶单体上的两个位点之间以及两个单体之间测量FRET（或淬灭）。 通过计数染料（每个单体一种染料）确定与每个DNA结合的解旋酶的数量。将在同时检测到的单分子信号之间进行关联。 例如，我们将测量与DNA结合的解旋酶单体的数量和DNA解旋，以确定SF1解旋酶的活性形式是单体还是二聚体。

We will study conformational changes of DNA helicases and functions using novel fluorescence assays. The assays include single molecule fluorescence resonance energy transfer (smFRET), quenching, single molecule counting, and ensemble FRET. Single molecule techniques can measure helicase activities in real time free from ensemble averaging. These techniques are also applicable to many biological systems, and indeed we have used them recently to detect conformational changes in RNA molecules and to study the folding pathways and catalysis of a ribozyme. The specific helicases will be from SF1 helicase family that includes e.coli Rep and UvrD helicases. The mechanistic information obtained should facilitate studies of other helicases as well. 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. Helicases also share many properties with other molecular motors. Hence a fundamental understanding of helicases is of both scientific and medical importance. In a previous study, we have used smFRET between dyess attached to DNA to study E. coli Rep helicase. We immobilized DNA on a polymer-coated surface, which enabled extended observation time while maintaining nearly complete biochemical activity. Unwinding of only a few base pairs, hence the distance change between two dyes attached to the DNA could be detected via smFRET. We also discovered a number of new conformations and determined the fluctuation rates among them. Further work using these techniques, proposed here, is poised to answer many fundamental questions. (1) Is oligomerization of helicase necessary for DNA unwinding and if so why? (2) How many base pairs are unwound per biochemical cycle and what factors influence the unwinding processivity? How tightly coupled is ATP hydrolysis to unwinding? (3) What are the functional roles of helicase conformational change; how nucleotide and DNA binding influence them? (4) What is the origin for directionality of DNA unwinding? To achieve these goals, we will use both ensemble and single molecule measurements of dyes attached to various sites on DNA and helicase. Specifically, FRET (or quenching) will be measured between sites on DNA, between helicase and DNA, between helicase and ATP, between two sites on helicase monomer, and between two monomers. The number of helicases bound to each DNA will be determined by counting the dyes (one dye per monomer). Correlation will be made between single molecule signals detected simultaneously. For example, we will measure both the number of helicase monomers bound to DNA and DNA unwinding to determine if the active form of SF1 helicase is monomer or dimer.