Single molecule nucleic acid enzymology

单分子核酸酶学

• 批准号: 7359611
• 负责人: Kevin Douglas Raney
• 金额: $ 32.07万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 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/7359611
• 关键词: 2' ,3' -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

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解旋酶实际上是DNA代谢的各个方面所必需的，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鉴定出来。诸如Xeroderma Commentosum，Cockayne's综合征，Bloom's综合征和Werner's综合征等疾病与DNA解旋酶编码的特定基因的缺陷有关。对这些基本生化过程的全面描述需要对解旋酶机制的详细了解。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。在NS3H解旋酶的情况下，该机制称为DDA解旋酶或功能合作模型的合作Inch虫模型。人们认为每个旋转酶都通过英寸虫的易位模式起作用，但是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。该R24研究建议的目的是将研究人员汇集在一起​​，以开发可以在单分子水平上直接可视化解旋酶移动的方法，以测试合作的Inch虫机制，并为研究DNA上的多蛋白络合物奠定基础。解旋酶将用高荧光纳米晶体标记，从而使人或一组分子直接观察到。在解开双链DNA（特定的目标1）和单链DNA上的易位（特定目标2）时，将单个和多个解旋酶直接可视化。将检查具有独特特性的三种解旋酶。 TRAL是一种高度的过程，而DDA和NS3H是过程中较差的解旋酶。提议DDA和NS3H合作发挥作用，而TRAL则提议充当单体。该研究建议是对计划公告PA 03-127的直接回应，即“研究和协作方法的研究方法”，这是为了促进使用NIH资助的研究人员之间使用不同技术来研究常见问题的研究人员之间的协作。