DNA helicases and associated factors in genome stability

DNA 解旋酶和基因组稳定性的相关因素

• 批准号: 10388769
• 负责人: Matthew Linne Bochman
• 金额: $ 5.91万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388769
• 关键词: Alleles; Animal Model; Biochemical; Biological Assay; Biological Models; Chemistry; Clinic; Custom; DNA; DNA Interstrand Crosslinking; DNA Repair; DNA biosynthesis; Data; Development; Digit structure; Disease; Equipment; Family; Fluorescence; Functional disorder; Funding; Gel; Genes; Genetic; Genome; Genome Stability; Goals; Helicase Gene; Homidium Bromide; Human; Image; In Vitro; Isotopes; Knowledge; Label; Lead; Link; Maintenance; Mass Spectrum Analysis; Membrane; Mutate; Mutation; Nucleic Acids; Organ; Patients; Proteins; Radioisotopes; Research; Role; Sampling; Scanning; Sepharose; Slide; Stains; Techniques; Telomere Maintenance; Time; Transillumination; Work; base; crosslink; experimental study; genome integrity; helicase; imager; imaging system; in vivo; insight; mutant; next generation sequencing; protein expression; protein protein interaction; recombinational repair; repaired; therapeutic target; ultraviolet; virtual

PROJECT SUMMARY DNA helicases function in virtually all aspects of DNA replication, recombination, and repair. As such, they are vital to maintaining genome integrity and are disease linked when mutated. Despite many in vivo and in vitro advances in working with helicases, there is a gap in knowledge connecting mutant alleles of helicase genes to the treatment of patients in clinics. The objective of my research is to gain mechanistic insight into how DNA helicases function in genome maintenance and why their dysfunction leads to disease. Toward this goal, we are studying PIF1 and RecQ family helicases, which are evolutionarily conserved and because mutations in the human genes encoding these helicases are associated with multiple diseases. Our current work focuses on the roles of RecQ helicases in DNA inter-strand crosslink (ICL) repair and RecQ and Pif1 helicases in telomere maintenance. To perform this work, we will employ a variety of classic and cutting edge experimental techniques, from standard in vitro enzymatic assays and model organism genetics to next- generation sequencing, crosslinking mass spectrometry, and the development of custom click chemistry probes. Overall, this work will provide fundamental data critical to understanding how PIF1 and RecQ family helicases aid in the maintenance of genome stability, and it will ultimately lead to therapeutic targets and treatments for helicase-linked diseases. I am requesting funds to purchase an Odyssey CLx Imaging System with a D-Digit attachment. This imager enables two-channel (700 and 800 nm) near-infrared fluorescence and chemiluminescent imaging of membranes, gels, plates, slides, and even large samples such as organs. The D-Digit further extends functionality to the scanning, imaging, and analysis of agarose DNA gels stained with ethidium bromide or a variety of safe stains (e.g., GelRed) without the need for ultraviolet transillumination. We routinely perform gel- based assays to assess protein expression, protein-protein interactions, protein-nucleic acid interactions, and the biochemical activities of DNA helicases and associated proteins, often using expensive radioisotopes for labeling. Further, multiple pieces of shared equipment spread across buildings on campus are necessary to visualize and analyze our experiments, and our time on the equipment is limited. The purchase of the Odyssey CLx and D-Digit will provide a dedicated, isotope-free solution to our gel-based imaging and analysis needs.

项目摘要 DNA解旋酶在DNA复制，重组和修复的几乎所有方面都起作用。像这样， 它们对于维持基因组完整性至关重要，并且在突变时疾病是关联的。尽管有很多体内和 在使用解旋酶时的体外进步，在连接解旋酶突变等位基因的知识上存在差距 诊所中患者治疗的基因。我研究的目的是获得机械洞察力 DNA解旋酶在基因组维持中的作用以及为什么其功能障碍导致疾病。走向这个 目标，我们正在研究PIF1和RECQ家族解旋酶，它们在进化上保守，因为 编码这些解旋酶的人类基因中的突变与多种疾病有关。我们的目前 工作重点介绍了RECQ解旋酶在DNA链间交叉链接（ICL）修复以及RECQ和PIF1中的作用 端粒维护中的解旋酶。为了执行这项工作，我们将采用各种经典和尖端 实验技术，从标准的体外酶试验和模型生物遗传学到接下来 生成测序，交联质谱和自定义点击化学的开发 探针。总体而言，这项工作将为了解PIF1和RECQ家庭如何提供基本数据 解旋酶有助于维持基因组稳定性，并最终导致治疗靶标和 解旋酶连接疾病的治疗方法。 我要求资金购买带有D数字附件的Odyssey CLX成像系统。这 成像仪可实现两通道（700 nm和800 nm）近红外荧光和化学成像的成像 膜，凝胶，板，幻灯片，甚至大型样品，例如器官。 D数字进一步扩展 用溴化乙锭或A染色的琼脂糖DNA凝胶的扫描，成像和分析的功能 各种安全污渍（例如，凝胶），无需紫外线透明。我们通常执行凝胶 - 基于评估蛋白质表达，蛋白质 - 蛋白质相互作用，蛋白核酸相互作用和 DNA解放酶和相关蛋白的生化活性，通常使用昂贵的放射性病 标签。此外，需要在校园内的建筑物中分布多种共享设备 可视化和分析我们的实验，我们在设备上的时间受到限制。购买奥德赛 CLX和D-Digit将为我们的基于凝胶的成像和分析需求提供专用的，无同位素的解决方案。