DNA helicases and associated factors in genome stability

DNA 解旋酶和基因组稳定性的相关因素

• 批准号: 10788644
• 负责人: Matthew Linne Bochman
• 金额: $ 22.24万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10788644
• 关键词: Alleles; Biochemical; Biological Assay; Biological Models; Biophysics; Chemistry; Chromatography; Clinic; Custom; DNA Interstrand Cross-Link Repair; DNA Repair; DNA biosynthesis; Data; Dedications; Development; Devices; Disease; Family; Functional disorder; Funding; Genes; Genetic; Genetic Recombination; Genome; Genome Stability; Goals; Helicase Gene; Human; In Vitro; Knowledge; Link; Maintenance; Mass Spectrum Analysis; Measurement; Molecular Sieve Chromatography; Mutate; Mutation; Patients; Physiological; Preparation; Process; Proteins; Recombinant Proteins; Research; Resolution; Role; System; Techniques; Telomere Maintenance; Work; crosslink; design; experimental study; genome integrity; helicase; improved; in vivo; insight; model organism; mutant; next generation sequencing; repaired; single molecule; therapeutic target; virtual

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.

项目概要 DNA 解旋酶几乎在 DNA 复制、重组和修复的所有方面发挥作用。像这样， 它们对于维持基因组完整性至关重要，并且在突变时与疾病相关。尽管许多体内和 解旋酶体外研究进展，连接解旋酶突变等位基因的知识存在空白 基因在诊所治疗患者。我研究的目的是获得机制上的洞察 DNA 解旋酶如何在基因组维护中发挥作用以及它们的功能障碍为何会导致疾病。朝这个方向 为了实现这一目标，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的突变与多种疾病相关。我们目前的 工作重点是 RecQ 解旋酶在 DNA 链间交联 (ICL) 修复以及 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前沿的技术 实验技术，从标准体外酶测定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开发 探针。总体而言，这项工作将提供对于理解 PIF1 和 RecQ 家族如何发挥作用至关重要的基础数据 解旋酶有助于维持基因组稳定性，最终将导致治疗靶点和 解旋酶相关疾病的治疗。 我正在请求资金购买 Refeyn TwoMP 质量光度计。该装置可实现快速质量 以单分子精度测量小体积的稀释生物分子。我们定期生成 用于生化测定的重组蛋白制剂，并了解寡聚状态和 生理条件下的单分散性对于我们的设计、分析和解释至关重要 实验。我们目前使用尺寸排阻色谱 (SEC) 来测定重组蛋白质量， 但 SEC 速度慢、分辨率低，并且需要大量非常浓缩的蛋白质。因此，美国证券交易委员会 限制了我们色谱系统的可用性，并且与无法纯化的蛋白质不兼容 大量或不能耐受浓缩过程。购买质量光度计将提供 使用廉价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所有的项目。