HELICASE-CATALYZED DNA UNWINDING

解旋酶催化的 DNA 解旋

• 批准号: 2183541
• 负责人: Timothy M Lohman
• 金额: $ 20.01万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183541
• 关键词: DNA; DNA binding protein; DNA replication origin; adenosine triphosphate; adenosinetriphosphatase; analytical ultracentrifugation; bioenergetics; chemical kinetics; chemical stability; conformation; dimer; electron microscopy; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescence polarization; gel electrophoresis; helicase; light scattering; nucleic acid denaturation; nucleic acid structure; oligonucleotides; polynucleotides; stop flow technique; synthetic nucleotide; thermodynamics

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.

拟议的研究旨在从分子上了解 解旋酶这类DNA结合蛋白 在ATP依赖性反应中，以 500-103碱基对/秒。 这类蛋白质是 E.大肠杆菌和可能所有 有机体 特别是E. coli rep基因产物（Rep）和E. 大肠杆菌uvrD基因产物（解旋酶II）将使用多种 生物化学和生物物理技术。 体外定量研究 纯化的蛋白质与自身的平衡结合，单个- 链和双链DNA，以及它们的核苷酸辅因子， 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 对于各种相互作用，可以用来获得热力学 信息，这是必要的，以了解基础的稳定性 这些复合物。 这些相互作用的动力学研究也将在 追求理解相互作用的机制和途径。 在 与这些研究平行的是，各种长的双链DNA的解旋， 将对底物进行定量检查，以获得有关 解旋的速率和过程。 用于螺旋展开 在解旋酶的作用下，蛋白质必须沿着DNA移位而不解离， 这是一个非常重要的过程，尽管我们目前 对分子机制知之甚少。 解旋酶催化的 将在不存在DNA合成的情况下检查DNA解旋反应， 因此提供了一个简单的系统来证明 反应 这些实验将用于评估f1的影响。 基因II蛋白对REp解旋反应的持续合成速率的影响， 以及螺旋不稳定蛋白的影响。 这些研究 专门针对理解解旋酶催化的DNA解旋 反应;然而，这也可能揭示热力学细节， 将增加我们对蛋白质稳定性基础的一般知识， DNA复合物。 此外，从所获得的机械信息 这些蛋白质-DNA相互作用的动力学也应该有助于我们 了解其他蛋白质，必须易位沿着DNA，以 功能（由ATP水解热驱动），例如，RNA和DNA 聚合酶 由于DNA复制是细胞生长的基础， 生物体，了解这样一个基本方面的机制 酶催化的DNA解旋无疑会对我们的研究产生影响。 了解复制功能障碍的疾病。