DNA Helicases: Mechanisms and Functions

描述（由申请人提供）：解旋酶是普遍存在的酶，参与DNA和RNA代谢的几乎每个方面。该项目的重点是这个家族中最大的一类酶，超家族1B（SF 1B）。有限的结构信息减缓了我们对这类酶的理解。SF 1B解旋酶将ATP水解与DNA解旋偶联，但这一过程中的限速步骤尚不清楚。已知特定的氨基酸基序与DNA接触，但这些基序的动态作用仅被推断。SF 1B解旋酶与其他蛋白质如单链结合蛋白相互作用，但这些相互作用的生物化学和生物学作用在很大程度上未得到解决。在我们的知识中，填补这些空白的重要性与解旋酶在DNA代谢中发挥的许多作用有关，包括复制，修复和重组。解旋酶活性的分子缺陷与许多以基因组不稳定性、过早衰老和癌症为特征的人类遗传疾病直接相关。因此，我们必须了解这些酶的机制，以了解分子水平的缺陷如何导致这种毁灭性的疾病。来自噬菌体T4的Dda解旋酶已用作SF 1B解旋酶的原型模型系统。Dda的新结构数据使我们提出了一个机械化学耦合机制，涉及的领域，包括标准的解旋酶基序沿着与新的结构域是未表征的。解旋酶测定和DNA足迹将用于测试这种机制。我们将确定DNA解旋过程中ATP水解的动力学机制，以确定该过程中的总体限速步骤，这是目前未知的。提出通过构象变化驱动解旋酶的蛋白质结构域将通过快速化学足迹法进行检查，该方法揭示DNA在活性位点内是紧密结合还是松散结合。高迁移率蛋白质基序将通过氢-氘交换来鉴定，以确定蛋白质结构和动力学之间的关系。解旋酶酶学中的一个主要未解问题涉及酶与每一条DNA链之间的相互作用。X射线晶体学、质谱和动力学方法的组合将用于鉴定酶表面上的所有DNA结合位点。这些新的DNA结合位点的结构-功能关系将通过DNA解旋实验来确定。解旋酶从DNA中去除蛋白质的机制将使用单分子方法进行研究。蛋白质-蛋白质相互作用的作用将通过产生解旋酶的拴系的二聚体形式并检查该酶置换DNA结合蛋白质的能力来确定。在这个建议中提出的问题的答案将推进该领域的深度（解旋酶酶学）和广度（解旋酶与蛋白质伴侣的相互作用），其中每一个将有助于理解这些酶在正常和致病的DNA代谢途径中发挥的作用。这项工作将提供实验和概念工具，以调查其他类别的解旋酶。

DESCRIPTION (provided by applicant): Helicases are ubiquitous enzymes involved in virtually every aspect of DNA and RNA metabolism. This project focuses on one of the largest classes of this family of enzymes, superfamily 1B (SF1B). Limited structural information has slowed progress of our understanding of this class of enzymes. SF1B helicases couple ATP hydrolysis to DNA unwinding, but the rate limiting steps in this process are unknown. Specific amino acid motifs are known to make contact with DNA, but the dynamic role of these motifs has only been inferred. SF1B helicases interact with other proteins such as single-stranded binding proteins, but the biochemical and biological roles of these interactions are largely unaddressed. The importance of filling in these gaps in our knowledge relates to the many roles that helicases play in DNA metabolism including replication, repair, and recombination. Molecular defects in helicase activity have been directly linked to numerous human genetic diseases characterized by genome instability, premature aging, and cancer. Therefore, it is critical that we understand the mechanisms of these enzymes in order to understand how defects at the molecular level can lead to such devastating diseases. Dda helicase from bacteriophage T4 has served as the prototypical model system for the SF1B helicases. New structural data for Dda has led us to propose a mechano-chemical coupling mechanism that involves domains that include the standard helicase motifs along with novel domains that are uncharacterized. Helicase assays and DNA footprinting will be used to test this mechanism. We will determine the kinetic mechanism for ATP hydrolysis during DNA unwinding to determine the overall rate-limiting step in the process, which is currently unknown. Protein domains that are proposed to drive the helicase through conformational changes will be examined by rapid chemical footprinting methods that reveal whether DNA is bound tightly or loosely within the active site. High mobility protein motifs will be identified by hydrogen-deuterium exchange in order to determine the relationship between protein structure and dynamics. One of the major unanswered questions in helicase enzymology relates to the interaction between the enzyme and each individual strand of DNA. A combination of x-ray crystallographic, mass spectrometric and kinetic approaches will be used to identify all of the DNA binding sites on the surface of the enzyme. The structure-function relationship of these novel DNA binding sites will be determined through DNA unwinding experiments. The mechanism by which helicases remove proteins from DNA will be investigated using single molecule approaches. The role of protein-protein interactions will be determined by creating a tethered, dimeric form of the helicase and examining the ability of this enzyme to displace DNA-bound proteins. Answers to the questions posed in this proposal will advance the field in depth (helicase enzymology) and breadth (helicase interactions with protein partners), each of which will facilitate understanding of the role that these enzymes play in normal and pathogenic pathways of DNA metabolism. This work will provide experimental and conceptual tools to investigate other classes of helicases.