DNA helicases and associated factors in genome stability

项目总结 DNA解旋酶几乎在DNA复制、重组和修复的各个方面发挥作用。因此， 它们对维持基因组的完整性至关重要，并且在突变时与疾病相关。尽管许多人在体内和 在体外处理解旋酶方面的进展，在连接解旋酶突变等位基因的知识上存在差距 基因能在临床上治疗病人。我研究的目的是从机械上洞察 DNA解旋酶如何在基因组维护中发挥作用，以及为什么它们的功能障碍会导致疾病。朝向这个方向 我们正在研究PIF1和RecQ家族解旋酶，它们在进化上是保守的，因为 编码这些解旋酶的人类基因突变与多种疾病有关。我们目前的情况 RecQ解旋酶在DNA链间交联(ICL)修复和RecQ和Pif1中的作用 端粒维持中的解旋酶。为了完成这项工作，我们将采用各种经典和尖端的 实验技术，从标准的体外酶分析和模型生物遗传学到下一步- 世代测序、交联质谱和定制点击化学的发展 探测器。总体而言，这项工作将提供对理解PIF1和RecQ家族如何 解旋酶有助于维持基因组的稳定性，它最终将导致治疗靶点和 解旋酶相关疾病的治疗。 我要求资金购买一个奥德赛CLX成像系统与D数字附件。这 Imager支持双通道(700和800 nm)近红外荧光和化学发光成像 膜、凝胶、平板、载玻片，甚至器官等大样本。D位数进一步扩展 用于扫描、成像和分析溴化乙锭染色的琼脂糖凝胶 多种安全染料(如GelRed)，无需紫外线透光。我们通常会做凝胶实验- 基于评估蛋白质表达、蛋白质-蛋白质相互作用、蛋白质-核酸相互作用以及 DNA解旋酶和相关蛋白质的生化活性，通常使用昂贵的放射性同位素来 贴标签。此外，在校园内的建筑物中分布多个共享设备是必要的 想象和分析我们的实验，我们在设备上的时间是有限的。收购《奥德赛》 CLX和D-Digit将为我们基于凝胶的成像和分析需求提供专用的、不含同位素的解决方案。

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.