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。 我请求资金购买一个四件套的Cerillo Stratus平板阅读器，带有适配器夹子，用于 振动器安装。这些板材读取器将能够连续实时监测高达 在四个96孔板中有384个独立的培养物。万能夹具允许安装层状物 任何商业平台摇摆器中的设备。我们经常进行比较增长曲线分析， 酿酒酵母菌株，以评估突变、小分子处理和/或 异源蛋白在细胞上的表达。这些分析目前是在Biotek Synergy平板上进行的 阅读器，但它们需要24到48小时的持续使用才能完成。因此，板阅读器是 不能用于其他目的(例如，DNA和蛋白质量化)，我的研究小组成员 提前几周报名以预定时间。购买Stratus Four Pack将提供专用车牌 用于增长曲线分析的读者，提高我们的吞吐量并支持我们的所有项目。

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 a four-pack of Cerillo Stratus plate readers with adapter clamps for shaker installation. These plate readers will enable the continuous real-time optical density monitoring of up to 384 independent cultures in four 96-well plates. The universal clamps allow the installation of the Stratus devices in any commercial platform shaker. We routinely perform comparative growth curve analyses of Saccharomyces cerevisiae strains to gauge the effects of mutations, small molecule treatments, and/or heterologous protein expression on the cells. These assays are currently performed in a Biotek Synergy plate reader, but they take between 24 and 48 h of constant use to complete. Therefore, the plate reader is unavailable for other purposes (e.g., DNA and protein quantification), and members of my research group have to sign up weeks in advance to reserve time. The purchase of the Stratus four pack will provide dedicated plate readers for growth curve analyses, improving our throughput and supporting all of our projects.