DNA helicases and associated factors in genome stability

DNA 解旋酶和基因组稳定性的相关因素

基本信息

批准号：
10810234
负责人：
Matthew Linne Bochman
金额：
$ 0.84万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10810234
关键词：
Alleles Binding Biological Assay Biological Models Biological Sciences Biomedical Research Chemistry Clinic Complement Custom DNA Interstrand Cross-Link Repair DNA Maintenance DNA Repair DNA biosynthesis Data Development Disease Family Functional disorder Funding G-Quartets Genes Genetic Genetic Recombination Genome Genome Stability Goals Group Meetings Helicase Gene Human In Vitro Indiana Knowledge Link Maintenance Mass Spectrum Analysis Molecular Molecular Biology Mutate Mutation Patients Productivity Research Research Project Grants Role Saccharomyces cerevisiae Structure Techniques Telomere Maintenance Testing Toxic effect Universities Work career cell type crosslink experience genome integrity helicase in vivo insight member model organism mutant nanobodies next generation sequencing overexpression posters repaired summer research symposium therapeutic target undergraduate research undergraduate research experience virtual yeast genetics

项目摘要

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.

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。它们对于维持基因组完整性至关重要，并且在体内和突变时与疾病相关。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差距我的研究目的是深入了解基因对临床患者治疗的影响。 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。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我们目前的多种疾病有关。工作重点是 RecQ 解旋酶在 DNA 链间交联 (ICL) 修复以及 RecQ 和 Pif1 中的作用为了完成这项工作，我们将采用各种经典和前沿的解旋酶。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总的来说，这项工作将提供对于理解 PIF1 和 RecQ 系列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。在此申请中，我请求资金支持摩根女士为期 10 周的研究经历 2023 年夏天，Roush 女士来到我的实验室。该项目的目的是让 Roush 女士获得分子生物学和酵母遗传学方面的研究经验支持她追求职业生涯的目标 Roush 女士的生物医学研究项目将检查 G-四链体 (G4) DNA 之间的联系。 Roush 女士的目标是培育缺乏稳定性和基因组完整性的酿酒酵母菌株。与 G4 结构解旋和维持有关的一种或多种 DNA 解旋酶的酶活性。然后将测试在这些菌株中过度表达 G4 结合和稳定纳米抗体的效果以进行比较 Roush 女士将探讨 G4 稳定化对含有完整解旋酶的野生型细胞的毒性。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海报 2023 年夏末本科生研究研讨会。我进一步希望她的暑假研究经验是积极的，她将一直是我实验室的一名富有成效的成员，直到她毕业。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Fanconi anemia-independent DNA inter-strand crosslink repair in eukaryotes.

DOI：
10.1016/j.pbiomolbio.2020.08.005
发表时间：
2020-12
期刊：
Progress in biophysics and molecular biology
影响因子：
3.8
作者：
Rogers CM;Simmons Iii RH;Fluhler Thornburg GE;Buehler NJ;Bochman ML
通讯作者：
Bochman ML

Pif1 Activity is Modulated by DNA Sequence and Structure.

DOI：
10.1021/acs.biochem.1c00614
发表时间：
2022-01-04
期刊：
Biochemistry
影响因子：
2.9
作者：
Nickens DG;Bochman ML
通讯作者：
Bochman ML

Characterization of the telomerase modulating activities of yeast DNA helicases.

DOI：
10.1016/bs.mie.2021.08.005
发表时间：
2021
期刊：
Methods in enzymology
影响因子：
0
作者：
Nickens DG;Bochman ML
通讯作者：
Bochman ML

Overcoming stochastic variations in culture variables to quantify and compare growth curve data.

DOI：
10.1002/bies.202100108
发表时间：
2021-08
期刊：
BioEssays : news and reviews in molecular, cellular and developmental biology
影响因子：
0
作者：
Sausen CW;Bochman ML
通讯作者：
Bochman ML

Lysine acetylation regulates the activity of nuclear Pif1