DNA helicases and associated factors in genome stability

项目摘要 DNA解旋酶几乎在DNA复制、重组和修复的所有方面发挥作用。因此，在本发明中， 它们对维持基因组完整性至关重要，突变时与疾病有关。尽管许多体内和 在体外研究解旋酶的进展中，连接解旋酶突变等位基因的知识存在空白 基因在临床上的应用我研究的目的是从机理上了解 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。朝着这个 我们正在研究PIF 1和RecQ家族解旋酶，它们在进化上是保守的， 编码这些解旋酶的人类基因中的突变与多种疾病有关。我们目前 工作重点是RecQ解旋酶在DNA链间交联（ICL）修复中的作用，以及RecQ和Pif 1 端粒维持中的解旋酶。为了完成这项工作，我们将聘请各种经典和尖端的 实验技术，从标准的体外酶测定和模式生物遗传学到下一个- 代测序、交联质谱和定制点击化学的发展 probes.总的来说，这项工作将提供关键的基本数据，以了解PIF 1和RecQ家族 解旋酶有助于维持基因组的稳定性，并且它将最终导致治疗靶点， 解旋酶相关疾病的治疗。 我申请资金购买Odyssey CLx成像系统（带D位附件）。这 成像仪能够实现双通道（700和800 nm）近红外荧光和荧光发光成像， 膜、凝胶、板、载玻片，甚至大的样品如器官。D数字进一步扩展 功能性的扫描，成像，和分析琼脂糖DNA凝胶染色溴化乙锭或 各种安全污渍（例如，GelRed），而不需要紫外线透照。我们通常做凝胶- 基于分析来评估蛋白质表达、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物化学活性，通常使用昂贵的放射性同位素 标签。此外，校园内建筑物之间分布的多件共享设备是必要的， 可视化和分析我们的实验，我们在设备上的时间是有限的。购买奥德赛 CLx和D-Digit将为我们的凝胶成像和分析需求提供专用的无同位素解决方案。

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.