SSB Protein/DNA Interactions

SSB 蛋白质/DNA 相互作用

• 批准号: 7616850
• 负责人: Timothy M Lohman
• 金额: $ 40.39万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616850
• 关键词: Affect; Affinity; Antibiotics; Bacterial Proteins; Binding; Binding Proteins; C-terminal; Calorimetry; Charge; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Polymerase III; DNA Repair; DNA biosynthesis; Defect; Development; Differential Scanning Calorimetry; Dissociation; Enzymes; Equilibrium; Escherichia coli; Eukaryota; Fluorescence; Heating; Homo; Human; Individual; Ions; Kinetics; Knowledge; Lead; Link; Malignant Neoplasms; Methods; Molecular; Movement; Nucleic Acids; Optics; Pathway interactions; Play; Premature aging syndrome; Process; Property; Protein Subunits; Proteins; RNA polymerase sigma 54; Research; Role; SS DNA BP; Single-Stranded DNA; Site; Sodium Chloride; Specificity; Spectrum Analysis; System; Tail; Techniques; Temperature; Testing; Thermodynamics; Titrations; Yeasts; enthalpy; flexibility; human disease; in vivo; melting; novel; nucleic acid metabolism; recombinational repair; single molecule; single-molecule FRET

DESCRIPTION (provided by applicant): Single Stranded Binding (SSB) Proteins are central players in DNA replication, recombination and repair due to their interactions with DNA and myriad accessory proteins. We seek a molecular understanding of the interactions of the E. coli EcoSSB, and the human RPA proteins, with single stranded (ss) DNA and accessory proteins. Ensemble thermodynamic and kinetic approaches, as well as novel single molecule approaches will be used to investigate the energetics and dynamics of these interactions. EcoSSB serves as a paradigm for a growing number of similar proteins possessing multiple DNA binding domains, displaying a complex array of multiple ssDNA binding modes, multiple inter-tetramer positive cooperativities and a negative cooperativity for ssDNA binding within an individual tetramer that regulates the binding modes. These binding modes differ in the extent to which ssDNA is wrapped around the tetramer and we hypothesize that they are used selectively in different processes in vivo. Our extensive knowledge of the EcoSSB protein makes it one of the best systems for continued studies of the thermodynamic profile of a protein-ssDNA binding system. The thermodynamics of ssDNA binding will be examined by isothermal titration calorimetry (ITC) and Differential Scanning Calorimetry (DSC), focusing on the contributions of protein conformational changes to the extremely large ?Hobs and the dramatic changes in ?CP,obs on salt concentration and temperature. A new major focus is to understand the dynamics of spontaneous wrapping/unwrapping of ssDNA around the SSB tetramer and to test for possible protein movement along ssDNA by a "rolling" mechanism using single molecule fluorescence techniques, Fluorescence Correlation Spectroscopy (PCS) and stopped-flow. Optical tweezer approaches will be used to examine the effects of the different SSB binding modes on RecA binding to SSB-coated ssDNA. Our studies of the RPA protein have shown that it also binds ssDNA in multiple binding modes and we will continue these studies. The effects of the different SSB-ssDNA modes on the specificity of EcoSSB binding to a variety of replication proteins will also be studied, as well as the kinetic mechanism of duplex DNA melting by these SSB proteins.

描述（由申请人提供）：由于与DNA和无数辅助蛋白的相互作用，单链结合（SSB）蛋白是DNA复制，重组和修复的核心参与者。我们寻求对大肠杆菌ECOSSB和人RPA蛋白的相互作用的分子理解，该蛋白具有单链（SS）DNA和辅助蛋白。集合热力学和动力学方法以及新型的单分子方法将用于研究这些相互作用的能量和动力学。 ECOSSB是具有多个具有多个DNA结合结构域的越来越多的类似蛋白的范式，显示了多个SSDNA结合模式的复杂阵列，多个四聚体阳性合作率和在单个Tetramer内的SSDNA结合的负合作性，从而调节结合模式。这些结合模式在ssDNA周围包裹的程度上有所不同，我们假设它们在体内有选择地用于不同过程。我们对ECOSSB蛋白的广泛了解使其成为持续研究蛋白质-SSDNA结合系统热力学谱的最佳系统之一。 ssDNA结合的热力学将通过等温滴定热量法（ITC）和差扫描量热法（DSC）检查，重点是蛋白质构象变化对极大的HOB？hobs的贡献，以及对？CP的巨大变化，对盐浓度和温度进行观察。一个新的主要重点是了解SSB四聚体周围ssDNA的自发包装/解开的动力学，并通过使用单分子荧光技术，荧光相关光谱（PC）和停止流量测试“滚动”机制沿SSDNA进行蛋白质运动。光学镊子方法将用于检查不同SSB结合模式对RECA结合与SSB涂层ssDNA的影响。我们对RPA蛋白的研究表明，它也以多种结合模式结合ssDNA，我们将继续这些研究。还将研究不同SSB-SSDNA模式对ECOSSB结合与各种复制蛋白的特异性的影响，以及这些SSB蛋白的双链DNA融化的动力学机制。