Single molecule nucleic acid enzymology

单分子核酸酶学

基本信息

批准号：
7247451
负责人：
Kevin Douglas Raney
金额：
$ 34.01万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7247451
关键词：
2&apos ,3&apos -Dideoxyadenosine Attention Bacteriophage T4 Binding Binding Proteins Biochemical Process Biological Assay Biotin Bloom Syndrome Cockayne Syndrome Code Collaborations DNA Defect Disease Ensure Enzymatic Biochemistry Enzymes Eukaryota Eukaryotic Cell Exhibits Filament Fluorescence Fluorescence Spectroscopy Funding Future Genes Genetic Transcription Goals Imagery Individual Label Link Localized Measures Metabolism Methodology Methods Modeling Monoclonal Antibody R24 Movement Multiprotein Complexes NIH Program Announcements Nucleic Acids Numbers Procedures Prokaryotic Cells Property Proteins Quantum Dots RNA RNA replication Research Research Personnel Research Proposals Resolution Single-Stranded DNA Site Spectrum Analysis Staging Streptavidin System Technology Testing Thinking Travel Virus Werner Syndrome Xeroderma Pigmentosum ds-DNA helicase monomer nanocrystal protein protein interaction recombinase recombinational repair research study response single molecule success

项目摘要

DESCRIPTION (provided by applicant): DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. These enzymes are ubiquitous, having been identified in viruses, prokaryotes, and eukaryotes. Diseases such as xeroderma pigmentosum, Cockayne's syndrome, Bloom's syndrome, and Werner's syndrome, have been linked to defects in specific genes coding for DNA helicases. A comprehensive description of these essential biochemical processes requires detailed understanding of helicase mechanisms. For some helicases, enzymatic activity is dependent on the number of helicase molecules that are bound onto the DNA. This mechanism is referred to as the cooperative inchworm model n the case of Dda helicase or the functional cooperativity model in the case of NS3h helicase. Each individual helicase is thought to function via an inchworm mode of translocation, but multiple molecules bound to a single substrate exhibit greater activity. Cooperativity can result through structural or functional interactions. The goal of this R24 research proposal is to bring together investigators to develop methods that will allow direct visualization of helicase movement at the single molecule level in order to test the cooperative inchworm mechanism and lay the groundwork for studying multiprotein complexes on DNA. Helicases will be labeled with highly fluorescent nanocrystals, thereby enabling individual or groups of molecules to be observed directly. Direct visualization of single and multiple helicases during unwinding of double stranded DNA (specific aim 1) and translocation on single stranded DNA (specific aim 2) willl be measured. Three helicases that have unique properties will be examined. Tral is a highly processive helicase whereas Dda and NS3h are poorly processive helicases. Dda and NS3h are proposed to function cooperatively whereas Tral is proposed to function as a monomer. This research proposal is a direct response to the program announcement PA 03-127, "INTEGRATIVE AND COLLABORATIVE APPROACHES TO RESEARCH" which is to facilitate collaboration between NIH-funded investigators who use different technologies to study a common problem.

描述（由申请人提供）：DNA 代谢的几乎每个方面都需要 DNA 解旋酶，包括复制、修复、重组和转录。这些酶无处不在，已在病毒、原核生物和真核生物中得到鉴定。着色性干皮病、科凯恩综合征、布卢姆综合征和维尔纳综合征等疾病与编码 DNA 解旋酶的特定基因缺陷有关。对这些基本生化过程的全面描述需要详细了解解旋酶机制。对于某些解旋酶，酶活性取决于结合到 DNA 上的解旋酶分子的数量。该机制在 Dda 解旋酶的情况下被称为协同尺蠖模型，在 NS3h 解旋酶的情况下被称为功能协同模型。每个单独的解旋酶被认为通过尺蠖易位模式发挥作用，但与单个底物结合的多个分子表现出更大的活性。合作性可以通过结构或功能相互作用产生。 R24 研究计划的目标是将研究人员聚集在一起，开发能够在单分子水平上直接可视化解旋酶运动的方法，以测试尺蠖的协作机制，并为研究 DNA 上的多蛋白复合物奠定基础。解旋酶将用高荧光纳米晶体标记，从而能够直接观察单个或一组分子。将测量双链 DNA 解旋（具体目标 1）和单链 DNA 易位（具体目标 2）过程中单个和多个解旋酶的直接可视化。将检查三种具有独特性质的解旋酶。 Tral 是一种高度进行性的解旋酶，而 Dda 和 NS3h 是一种进行性较差的解旋酶。 Dda 和 NS3h 被建议协同发挥作用，而 Tral 被建议作为单体发挥作用。该研究提案是对计划公告 PA 03-127“综合和协作研究方法”的直接回应，该计划旨在促进 NIH 资助的研究人员之间的合作，这些研究人员使用不同的技术来研究共同问题。