DNA Unwinding and Translocation by Helicases

描述(由申请人提供)： DNA解旋酶几乎是DNA新陈代谢的各个方面所必需的，包括复制、修复、重组和转录。要全面了解这些基本的生化过程，就需要详细了解解旋酶的机制。我们正在研究来自噬菌体T4的DDA解旋酶，作为超家族(SF)1解旋酶的代表，这是这类酶中最大的一类。在之前的资助周期中，我们测试了这样一个假设，即DDA解开双链(DS)DNA在很大程度上是单链(SS)DNA单向易位的结果。我们的结果支持了我们的假设，数据与解旋酶活性的“尺虫模型”一致。关于“尺寸虫”的确切功能，在细节上有几个不同之处，目前的具体目标就是在这个背景下设计的。我们目前的DDA函数模型可能解释了一些差异。我们认为，DDA可以作为一个单体发挥作用，但多个单体可以协同作用，促进移位和解离。我们将这种新的模型称为合作尺寸虫模型。协同作用在该机制中的作用可能是减少当解旋酶遇到双链DNA或DNA结合蛋白等易位挑战时发生的滑移。在当前的周期中，我们建议测试这一新的假设，并在我们继续专注于DDA的同时扩展项目的目标。我们将确定单体和多聚体DDA的DNA解离的动力学步长。我们将测量在预稳定状态条件下和在过量酶存在的情况下的ATP水解量。DNA解离的加工性将作为与底物结合的DDA分子数量的函数来研究。DNA与DDA的相互作用将用交联质谱联用来研究。将继续进行结晶学和结构建模研究，以将解旋酶的结构与生化功能联系起来。最后，将开发新的方法在单分子实验中直接观察解旋酶的移位和解离。

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.