HELICASE - CATALYZED DNA UNWINDING

解旋酶 - 催化 DNA 解旋

• 批准号: 3305442
• 负责人: Timothy M Lohman
• 金额: $ 20.65万
• 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/3305442
• 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; light scattering; nucleic acid denaturation; nucleic acid structure; oligonucleotides; polynucleotides; stop flow technique; synthetic nucleotide; thermodynamics; 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; 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.

拟议的研究旨在获得对 DNA结合蛋白称为解旋酶的机制 以ATP依赖性反应的催化催化双链DNA，以 500-103基对/秒。 这类蛋白质是需要的 大肠杆菌的复制，重组和修复过程，可能是所有 有机体。 特别是大肠杆菌REP基因产物（REP）和E. 将使用多种 生化和生物物理技术。 体外定量研究 纯化蛋白与自身的平衡结合，单个 将滞留和双链DNA及其核苷酸辅因子进行 作为溶液变量的函数（温度，pH，单价盐， MG2+）。 这些变量对平衡结合常数的影响 因为各种相互作用可用于获得热力学 信息，这是了解稳定性基础所必需的 这些复合物。 这些相互作用的动力学研究也将是 追求了解相互作用的机制和途径。 在 与这些研究平行，解开各种长的双链DNA 将对基材进行定量检查，以获取有关 放松的费率和过程。 进行过程放松 解旋酶，蛋白质必须沿着DNA转运而不会解离，a 尽管我们目前 了解分子机制几乎没有理解。 解旋酶催化 在没有DNA合成的情况下，将检查DNA解开反应， 因此，提供了一个简单的系统，以证明 反应。 这些实验将用于评估F1的影响 基因II蛋白关于REP放松反应的加工速率， 以及螺旋破坏蛋白质的影响。 这些研究是 专门用于了解解旋酶催化的DNA放松 反应；但是，这些可能还会揭示热力学细节 将提高我们对蛋白质稳定性基础的一般知识 DNA复合物。 此外，从 这些蛋白DNA相互作用的动力学也应有助于我们 了解必须沿DNA转移的其他蛋白质才能 功能（由ATP水解热驱动），例如RNA和DNA 聚合酶。 由于DNA复制对于所有人的细胞生长至关重要 有机体，对这样的基本方面的理解 酶催化的DNA的放松无疑会对我们的 了解复制故障的疾病。