Single Molecule Study of Helicase Mechanisms

我们将研究DNA解旋酶的构象变化和功能使用新的荧光分析。检测包括单分子荧光共振能量转移（smFRET），淬火，单分子计数，和集合FRET。单分子技术可以实时测量解旋酶活性，而不需要集合平均。这些技术也适用于许多生物系统，事实上，我们最近已经用它们来检测RNA分子的构象变化，研究核糖酶的折叠途径和催化作用。特异解旋酶将来自SF1解旋酶家族，包括大肠杆菌Rep和UvrD解旋酶。所获得的机制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绕和易位结合起来的分子马达。许多生物体编码多种解旋酶，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至关重要。一些人类遗传疾病也与DNA解旋酶的突变有关。解旋酶还与其他分子马达具有许多相同的性质。因此，对解旋酶的基本理解具有科学和医学上的重要性。在之前的研究中，我们利用附着在DNA上的染料之间的smFRET来研究大肠杆菌的Rep解旋酶。我们将DNA固定在聚合物包覆的表面上，这样可以延长观察时间，同时保持几乎完全的生化活性。由于只有少数碱基对解绕，因此可以通过smFRET检测到附着在DNA上的两种染料之间的距离变化。我们还发现了一些新的构象，并确定了它们之间的波动率。在这里提出的使用这些技术的进一步工作将回答许多基本问题。解旋酶的寡聚化对DNA解绕是必要的吗？如果是，为什么？(2)每个生化循环解绕多少碱基对，解绕速率受哪些因素影响？ATP水解与解绕的耦合有多紧密？(3)解旋酶构象变化的功能作用；核苷酸和DNA结合是如何影响它们的？(4) DNA解绕的方向性的起源是什么？为了实现这些目标，我们将对附着在DNA和解旋酶不同位点上的染料进行整体和单分子测量。具体来说，将测量DNA位点之间、解旋酶与DNA之间、解旋酶与ATP之间、解旋酶单体上的两个位点之间以及两个单体之间的FRET（或猝灭）。与每个DNA结合的解旋酶的数量将通过计算染料（每个单体一个染料）来确定。将同时检测到的单分子信号进行关联。例如，我们将测量与DNA结合的解旋酶单体和DNA解绕的数量，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.