DNA Helicases: Mechanisms and Functions

描述(申请人提供)：解旋酶是普遍存在的酶，几乎涉及DNA和RNA新陈代谢的各个方面。这个项目的重点是这个酶家族中最大的类别之一，超家族1B(SF1B)。有限的结构信息减缓了我们对这类酶的理解进展。SF1B解旋酶将ATP水解与DNA解离结合起来，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还只是被推断出来。SF1B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化和生物学作用在很大程度上还没有被解决。填补这些知识空白的重要性与解旋酶在DNA新陈代谢中扮演的许多角色有关，包括复制、修复和重组。解旋酶活性的分子缺陷与许多人类遗传性疾病直接相关，这些疾病的特征是基因组不稳定、过早衰老和癌症。因此，为了了解分子水平上的缺陷如何导致这种毁灭性的疾病，我们了解这些酶的机制是至关重要的。噬菌体T4的DDA解旋酶是SF1B解旋酶的原型模型系统。新的结构数据使我们提出了一种机械力-化学耦合机制，其中包括包括标准解旋酶基序的结构域以及未表征的新结构域。解旋酶分析和DNA足迹将被用来测试这一机制。我们将确定DNA解链过程中ATP水解的动力学机制，以确定该过程中的总体限速步骤，目前尚不清楚。被认为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方法进行检测，以揭示DNA在活性部位内是紧密结合还是松散结合。为了确定蛋白质结构和动力学之间的关系，将通过氢-氚交换来鉴定高流动性的蛋白质基序。解旋酶酶学中的一个主要未解答的问题与酶和DNA的每一条链之间的相互作用有关。将使用X射线结晶学、质谱学和动力学方法相结合的方法来鉴定酶表面的所有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解离实验来确定。解旋酶从DNA中去除蛋白质的机制将用单分子方法进行研究。蛋白质相互作用的作用将通过创建一种被拴住的二聚体解旋酶形式并检测这种酶取代DNA结合蛋白的能力来确定。对这一提议中提出的问题的回答将在深度(解旋酶酶学)和广度(解旋酶与蛋白质伙伴的相互作用)方面取得进展，每一项都将有助于理解这些酶在DNA代谢的正常和致病途径中所起的作用。这项工作将为研究其他类型的解旋酶提供实验和概念上的工具。

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.