DNA UNWINDING: THE ROLE OF E. COLI HELICASES AND SSB

DNA 解旋：大肠杆菌解旋酶和 SSB 的作用

• 批准号: 3278286
• 负责人: Timothy M Lohman
• 金额: $ 3.42万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278286
• 关键词: DNA binding protein; DNA replication; Escherichia coli; acidity /alkalinity; adenosine triphosphate; affinity chromatography; autoradiography; bacterial DNA; bacterial genetics; bacterial proteins; bacteriophage T4; binding proteins; centrifugation; chromatography; fluorescence microscopy; gel electrophoresis; genetic promoter element; genetic recombination; genetic transcription; magnesium; microorganism culture; molecular cloning; plasmids; protein metabolism; radiotracer; spermine; stop flow technique; thermodynamics

The proposed research is to conduct in vitro studies of the mechanisms by which duplex DNA is catalytically unwound by two classes of DNA binding proteins: helicases and helix destabilizing proteins (HDP). In particular, the E. coli coded Rep and Helicase II proteins (helicases) and the E. coli coded Single Strand Binding protein, SSB, (HDP) will be studied. The helicases catalyze the ATP dependent, unidirectional unwinding of duplex DNA, a process which requires translocation of the protein along DNA and is fundamental to DNA replication. Physical-biochemical techniques will be used to quantitatively investigate the equilibrium and kinetic binding properties of the purified proteins to DNA as a function of solution variables, with particular emphasis on the effects of low molecular weight ions (NaC1, MgC12, spermidine). The basis for the cooperativity in SSB-single stranded DNA binding and its dramatic dependence on salt concentration will also be studied. In parallel with these studies, the mechanisms of DNA unwinding catalyzed by Rep and Helicase II, and the role of SSB in this process, will be investigated, focusing on the rates, processivity, and role of ATP. The molecular aspects of the translocation of protein along DNA, which is fundamental to many cellular processes, will also be probed. The methods used to study the protein-DNA interactions include analytical sedimentaiton, gel electrophoresis, fluorescence and absorbance spectroscopy, affinity and gel chromatography and stopped-flow kinetics. The DNA unwinding reaction will be studied in the absence of DNA synthesis and other replication proteins, hence providing a simple system to probe the molecular aspects of unwinding and tranlocation. These studies are specifically directed to understand the helicase catalyzed DNA unwinding reaction and the mechanism of the ATP driven helicase translocation, however they will also reveal thermodynamic details which will increase our general knowledge of the factors which stabilize protein-nucleic acid interactions. Furthermore, the mechanistic information obtained from studies of the kinetics of these protein-DNA interactions should be useful in studies of other proteins which must also translocate along DNA (driven thermally or by hydrolysis of ATP); e.g. DNA and RNA polymerases. Since DNA replication is fundamental to cell growth, an understanding of its detals will undoubtedly increase our chances for determining how it malfunctions in diseases such as cancer.

拟议的研究是通过对机制进行体外研究 哪种双链DNA被两类DNA结合催化解开 蛋白质：动力稳定蛋白（HDP）。 在 特别是大肠杆菌编码的REP和Helicase II蛋白（解旋酶）和 大肠杆菌编码的单链结合蛋白SSB（HDP）将是 研究。 解旋酶催化ATP依赖性，单向 放松双工DNA，需要易位的过程 沿着DNA的蛋白质，是DNA复制的基础。 物理生物化学技术将用于定量研究 纯化蛋白与 DNA作为溶液变量的函数，特别强调 低分子量离子（NAC1，MGC12，精子）的影响。 基础 为了在ssb-single滞留的DNA结合及其戏剧性的同时性 还将研究对盐浓度的依赖性。 并联 这些研究，rep催化的DNA的机制和 解旋酶II以及SSB在此过程中的作用将被研究， 专注于ATP的速度，加工性和作用。 分子 蛋白质沿DN​​A易位的各个方面，这对于 也将探测许多细胞过程。 用于研究的方法 蛋白质-DNA相互作用包括分析stimentaiton，凝胶 电泳，荧光和吸光度光谱，亲和力和凝胶 色谱和停止流动动力学。 DNA解开反应将 在没有DNA合成和其他复制蛋白的情况下研究 因此，提供一个简单的系统来探测放松的分子方面 和三位一体。 这些研究专门用于理解 解旋酶催化DNA解开反应和ATP的机制 驱动的解旋酶易位，但是它们还将揭示热力学 细节将增加我们对因素的一般知识 稳定蛋白质核酸相互作用。 此外，机理 从这些蛋白-DNA的动力学研究中获得的信息 相互作用应在对其他蛋白质的研究中有用 沿着DNA转移（热驱动或通过ATP的水解）；例如脱氧核糖核酸 和RNA聚合酶。 由于DNA复制是细胞生长的基础 对它的肿瘤的理解无疑会增加我们的机会 确定IT在癌症等疾病中的故障。