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: 10447062
• 负责人: Matthew Linne Bochman
• 金额: $ 38.81万
• 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/10447062
• 关键词: Acetylation; Alleles; Animal Model; Biochemistry; Biological Assay; Biological Models; Biology; Chemistry; Clinic; Custom; DNA Interstrand Crosslinking; DNA Repair; DNA biosynthesis; Data; Development; Disease; Enzymes; Family; Fanconi' s Anemia; Functional disorder; Genes; Genetic; Genome; Genome Stability; Goals; Helicase Gene; Holoenzymes; Human; In Vitro; Knowledge; Lead; Life; Link; Lysine; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mutate; Mutation; Oncogenes; Pathway interactions; Patients; Predisposition; Premature aging syndrome; RECQL4 gene; Research; Role; Techniques; Telomerase; Telomere Maintenance; Telomere-Binding Proteins; Work; Yeasts; crosslink; genome integrity; helicase; in vivo; insight; mutant; next generation sequencing; nuclease; recombinational repair; reconstitution; repaired; therapeutic target; virtual

PROJECT SUMMARY DNA helicases represent one of very few enzyme classes that function in virtually all aspects of DNA replication, recombination, repair, and telomere maintenance. As such, they are vital to maintaining genome integrity and are disease linked when mutated. Thus, there is a critical need to comprehensively understand helicase biology and how these enzymes support genome integrity. 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, both because they are evolutionarily conserved in all domains of life (giving us our pick of model systems to dissect the various aspects of their biology) and because the human PIF1 and RecQ helicases are oncogenes. Indeed, mutations in the genes encoding these helicases are associated with multiple diseases, as well as predispositions to cancers and premature aging. 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. The proposed work will: 1) determine how RecQ4 subfamily helicases and Pso2 family nucleases function in Fanconi anemia- independent DNA ICL repair, identify other factors involved in this repair pathway, and examine how disease alleles of RECQL4 perturb its genome maintenance functions at a mechanistic level; 2) define how PIF1 and RecQ helicases synergistically modulate telomerase activity, determine the impacts of other telomere binding proteins on the biochemistry of these helicases, and reconstitute the telomerase holoenzyme in vitro; and 3) determine the regulatory effects of lysine acetylation on PIF1 family helicases in yeast and humans and how this is linked to genome integrity. 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.

项目概要 DNA 解旋酶是极少数在 DNA 复制的几乎所有方面发挥作用的酶类之一， 重组、修复和端粒维护。因此，它们对于维持基因组完整性和 突变时是否与疾病相关。因此，迫切需要全面了解解旋酶生物学 以及这些酶如何支持基因组完整性。尽管在体内和体外研究方面取得了许多进展 解旋酶，在将解旋酶基因突变等位基因与患者治疗联系起来方面存在知识空白 在诊所。我的研究目的是深入了解 DNA 解旋酶如何在基因组中发挥作用 维护以及为什么它们的功能障碍会导致疾病。为了这个目标，我们正在研究PIF1和RecQ 家族解旋酶，这都是因为它们在生命的所有领域中在进化上都是保守的（让我们选择模型 系统来剖析其生物学的各个方面）并且因为人类 PIF1 和 RecQ 解旋酶是 癌基因。事实上，编码这些解旋酶的基因突变与多种疾病相关，例如 以及癌症和过早衰老的倾向。我们目前的工作重点是 RecQ 解旋酶的作用 DNA 链间交联 (ICL) 修复以及端粒维护中的 RecQ 和 Pif1 解旋酶。拟议的 工作将：1) 确定 RecQ4 亚家族解旋酶和 Pso2 家族核酸酶如何在范可尼贫血中发挥作用 - 独立的 DNA ICL 修复，确定参与此修复途径的其他因素，并检查疾病如何发生 RECQL4 的等位基因在机械水平上扰乱其基因组维护功能； 2) 定义PIF1和 RecQ 解旋酶协同调节端粒酶活性，确定其他端粒结合的影响 蛋白质对这些解旋酶生物化学的影响，并在体外重建端粒酶全酶；和 3) 确定赖氨酸乙酰化对酵母和人类 PIF1 家族解旋酶的调节作用以及如何调节 与基因组完整性有关。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、交联质谱分析以及定制点击化学探针的开发。 总的来说，这项工作将提供对于理解 PIF1 和 RecQ 家族解旋酶如何发挥作用至关重要的基础数据 有助于维持基因组稳定性，最终将导致治疗靶点和治疗方法 解旋酶相关疾病。