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。 我要求资金购买四包Cerillo Stratus板读取器与适配器夹， 振动筛安装这些读板器将能够连续实时监测高达 在4个96孔板中的384个独立培养物。通用夹具允许安装Stratus 任何商业平台振动器中的设备。我们常规进行比较生长曲线分析， 酿酒酵母菌株，以测量突变、小分子处理和/或 细胞上的异源蛋白表达。这些试验目前在Biotek Synergy板中进行 阅读器，但它们需要24至48小时的持续使用才能完成。因此，酶标仪 不可用于其它目的（例如，DNA和蛋白质定量），我的研究小组成员已经 提前几周报名以预留时间购买的Stratus四包将提供专用板材 阅读器进行增长曲线分析，提高我们的吞吐量，并支持我们所有的项目。

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.