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。 我正在申请购买Refeyn TwoMP质量光度计的资金。这一装置可以实现快速质量 测量小体积稀释生物分子，具有单分子精度。我们通常会生成 用于生化分析的重组蛋白制剂，并知道寡聚状态和 生理条件下的单分散性对于设计、分析和解释我们的 实验。我们目前使用尺寸排除层析(SEC)来确定重组蛋白质的质量， 但SEC速度慢，分辨率低，需要大量高度浓缩的蛋白质。因此，SEC 限制了我们的层析系统的可用性，并且与无法在 大量或不能容忍浓缩过程。购买质量光度计将提供 使用价格低廉且易于使用的专用设备快速、高分辨率地解决这些问题 使用，提高我们的生物物理能力，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 Refeyn TwoMP mass photometer. This device enables rapid mass measurements of small volumes of dilute biomolecules, with single-molecule precision. We routinely generate recombinant protein preparations for biochemical assays, and knowing the oligomeric state and monodispersity under physiological conditions is critical to the design, analysis, and interpretation of our experiments. We currently use size-exclusion chromatography (SEC) to determine recombinant protein mass, but SEC is slow, has low resolution, and requires large volumes of very concentrated protein. Therefore, SEC limits the availability of our chromatography system and is incompatible with proteins that cannot be purified in large amounts or do not tolerate the concentration process. The purchase of a mass photometer will provide a rapid and high-resolution solution to these problems with a dedicated device that is inexpensive and easy to use, improving our biophysical capabilities and supporting all of our projects.