DNA Unwinding and Translocation by Helicases

描述（由申请人提供）： DNA解旋酶几乎是DNA代谢的每个方面所必需的，包括复制、修复、重组和转录。全面了解这些基本的生化过程需要详细了解解旋酶的机制。我们正在研究的Dda解旋酶，从噬菌体T4，作为超家族（SF）1解旋酶，这是这些酶的最大类的代表。在上一个资助周期中，我们测试了Dda解旋双链DNA主要是单链DNA单向易位的结果的假设。我们的研究结果支持了我们的假设和数据是一致的解旋酶活性的“尺蠖模型”。关于“尺蠖”的确切运作方式，在细节上有几处差异，正是在这一背景下设计了目前的具体目标。我们目前的Dda函数模型可以解释一些差异。我们认为Dda可以作为一个单体发挥作用，但是，多个单体可以合作，以加强易位和解旋。我们称这种新模型为合作尺蠖模型。协同性在该机制中的作用可能是减少当解旋酶遇到易位挑战（例如双链DNA或DNA结合蛋白）时发生的滑移。在当前的周期中，我们建议测试这个新的假设，并在继续关注Dda的同时扩展项目的目标。我们将确定Dda的单体和多聚体形式的DNA解旋的动力学步长。我们将测量在预稳态条件下和存在过量酶的情况下水解的ATP的量。DNA解旋的持续性将被研究为与底物结合的Dda分子数量的函数。DNA与Dda的相互作用将通过交联与质谱联用来研究。将进行晶体学和结构建模研究，以将解旋酶的结构与生化功能联系起来。最后，将开发新的方法来观察解旋酶易位和解旋直接在单分子实验。

DESCRIPTION (provided by applicant): DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. A comprehensive understanding of these essential biochemical processes requires detailed understanding of the mechanism of helicases. We are studying the Dda helicase, from bacteriophage T4, as a representative of super family (SF) 1 helicases, which is the largest class of these enzymes. In the previous grant cycle, we tested the hypothesis that unwinding of double-stranded (ds) DNA by Dda is largely a consequence of unidirectional translocation on single-stranded (ss) DNA. Our results have supported our hypothesis and the data are consistent with an 'inchworm model' for helicase activity. There are several discrepancies in the details for exactly how the 'inchworm' functions, and it is within this context that the current specific aims have been designed. Our current model for Dda function may explain some of the discrepancies. We suggest that Dda can function as a monomer, however, multiple monomers can cooperate to enhance translocation and unwinding. We term this new model the cooperative inchworm model. The role of cooperativity in the mechanism may be to reduce slippage that occurs when the helicase encounters a challenge to translocation such as duplex DNA or a DNA-binding protein. In the current cycle, we propose to test this new hypothesis, as well as expand the goals of the project as we continue to focus on Dda. We will determine the kinetic step size for DNA unwinding for monomeric and multimeric forms of Dda. We will measure the quantity of ATP hydrolyzed under pre-steady state conditions and in the presence of excess enzyme. Processivity of DNA unwinding will be studied as a function of the number of Dda molecules bound to the substrate. The interaction of DNA with Dda will be investigated by crosslinking coupled with mass spectrometry. Crystallographic and structural modeling studies will be pursued to relate the structure of the helicase to the biochemical function. Lastly, new methods will be developed to observe helicase translocation and unwinding directly in single molecule experiments.