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。 在这份申请中，我申请资金支持摩根女士为期10周的研究体验 2023年夏天，Roush在我的实验室里。这个项目的目的是使卢什女士能够获得 在分子生物学和酵母遗传学方面的研究经验，以支持她追求职业生涯的目标 生物医学研究。Roush女士的研究项目将研究G-四链(G4)DNA之间的联系 稳定性和基因组完整性。Roush女士的目标是培育出缺乏 一个或多个DNA解旋酶的活性与G4结构的解离和维持有关。她 然后将测试在这些菌株中过度表达G4结合和稳定的纳米体的效果以进行比较 G4稳定化对含有完整解旋酶的野生型细胞的毒性。卢什女士将 参加并出席我们的每周小组会议，她将在印第安纳大学展示一张海报 2023年夏末本科生研究座谈会。我更希望她的夏天 研究经验是积极的，她将一直是我实验室的富有成效的成员，直到她毕业。

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. In this application, I am requesting funds to support a 10-week research experience for Ms. Morgan Roush in my lab during the summer of 2023. The purpose of this project is to enable Ms. Roush to gain research experience in molecular biology and yeast genetics to support her goals to pursue a career in biomedical research. Ms. Roush’s research project will examine the link between G-quadruplex (G4) DNA stability and genome integrity. Ms. Roush aims to generate Saccharomyces cerevisiae strains lacking the enzymatic activity of one or more DNA helicases implicated in G4 structure unwinding and maintenance. She will then test the effects of over-expressing a G4-binding and -stabilizing nanobody in these strains to compare the toxicity of G4 stabilization to wild-type cells containing a full complement of helicases. Ms. Roush will attend and present at our weekly group meeting, and she will present a poster in the Indiana University Undergraduate Research Symposium at the end of summer 2023. It is my further hope that her summer research experience is positive and that she will remain a productive member of my lab until she graduates.