HELICASE - CATALYZED DNA UNWINDING

这项拟议的研究旨在从分子水平上了解 一类被称为解旋酶的DNA结合蛋白的机制 在依赖三磷酸腺苷的反应中，以下列速率催化解开双链DNA 500-103个碱基对/秒。这类蛋白质是需要的 在大肠杆菌中的复制、重组和修复过程 有机体。尤其是大肠杆菌rep基因产物(Rep)和E. ColiUvrD基因产物(解旋酶II)将使用各种 生化和生物物理技术。体外定量研究， 纯化的蛋白质与自身的平衡结合，单一的- 单链和双链DNA，以及它们的核苷酸辅助因子 作为溶液变量(温度、pH、一价盐、 Mg2+)。这些变量对平衡结合常数的影响 因为各种相互作用都可以用来获得热力学 信息，这是理解稳定的基础所必需的 这些复合体中。对这些相互作用的动力学研究也将 寻求了解相互作用的机制和途径。在……里面 在这些研究的同时，各种长的、双链DNA的解开 将对底物进行定量检查，以获得有关 平仓的速度和过程。用于逐步解锁 解旋酶，蛋白质必须不解离地沿DNA转移，a 这一进程至关重要，尽管我们目前 对其分子机制知之甚少。解旋酶催化的 DNA解离反应将在没有DNA合成的情况下进行检查， 因此提供了一种简单的系统来证明分子细节 反应。这些实验将被用来评估F1的影响 基因II蛋白对Rep解离反应的处理速度， 以及螺旋不稳定蛋白的影响。这些研究是 专门针对了解解旋酶催化的DNA解离 反应；然而，遗嘱可能也揭示了热力学细节 将增加我们对蛋白质稳定性基础的一般知识- DNA复合体。此外，从计算机上获得的机械性信息 这些蛋白质-DNA相互作用的动力学也应该有助于我们的 了解其他蛋白质必须沿DNA移位才能 功能(由ATP的水解热驱动)，例如，RNA和DNA 聚合酶。因为DNA复制是所有细胞生长的基础 有机体，对机制这样一个基本方面的理解 酶催化的DNA解离无疑将对我们的 对复制失灵的疾病的理解。

the proposed research is designed to obtain a molecular understanding of the mechanisms by which the class of DNA binding proteins called helicases catalytically unwind duplex DNA, in a ATP-dependent reaction, at rates of 500-103 base pairs/sec. this class of proteins is required for replication, recombination and repair processes in E. coli and likely all organisms. In particular, the E. coli rep gene product (Rep) and the E. coli uvrD gene product (helicase II) will be examined using a variety of biochemical and biophysical techniques. Quantitative studies in vitro, of the equilibrium binding of the purified proteins with themselves, single- stranded and duplex DNA, and their nucleotide cofactors will be undertaken as a function of solution variables (temperature, pH, monovalent salt, Mg2+). The effects of these variables on the equilibrium binding constants for the various interactions can be used to obtain thermodynamic information, which is necessary to understand the basis for the stability of these complexes. Kinetic studies of these interactions will also be pursued to understand the mechanisms and pathways of the interactions. In parallel with these studies, the unwinding of various long, duplex DNA substrates will be examined quantitatively to obtain information about the rates and processivities of unwinding. For processively unwinding helicases, the protein must translocate along DNA without dissociating, a process which is of fundamental importance, although we currently understand little about the molecular mechanism. the helicase-catalyzed DNA unwinding reaction will be examined in the absence of DNA synthesis, hence providing a simple system to prove the molecular details of the reaction. These experiments will be used to assess the effects of the f1 gene II protein on the rates of processivity of the REp unwinding reaction, as well as the effects of helix destabilizing proteins. These studies are specifically directed to understand the helicase-catalyzed Dna unwinding reactions; however, the will likely also reveal thermodynamic details that will increase our general knowledge of the basis for stability of protein- DNA complexes. Furthermore, the mechanistic information obtained from the kinetics of these protein-DNA interactions should also aid our understanding of other proteins that must translocate along DNA in order to function (driven thermally by hydrolysis of ATP), e.g., RNA and DNA polymerases. Since DNA replication is fundamental to cell growth in all organisms, an understanding of such a fundamental aspect as the mechanism of enzyme-catalyzed DNA unwinding will undoubtedly have an impact on our understanding of diseases in which replication malfunctions.