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.

项目摘要 DNA解旋酶代表了几乎在DNA复制的各个方面起作用的极少数酶类之一， 重组，维修和端粒维护。因此，它们对于维持基因组完整性和 突变时是疾病连接的。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。尽管有许多体内和体外进展 解旋酶，将解旋酶基因的突变等位基因与患者的治疗相连的知识存在差距 在诊所。我的研究的目的是获得有关DNA解旋酶在基因组中的发挥作用的机械洞察力 维护以及为什么其功能障碍导致疾病。为了实现这一目标，我们正在研究PIF1和RECQ 家庭解旋酶，既是因为它们在生活的所有领域都在进化上保守（为我们提供了模型的选择 剖析其生物学各个方面的系统），因为人类PIF1和RECQ解旋酶是 癌基因。实际上，编码这些解旋酶的基因中的突变与多种疾病有关，如 以及对癌症和过早衰老的倾向。我们目前的工作着重于RECQ解旋酶的角色 在DNA间交叉链接（ICL）修复中，端粒维护中的RECQ和PIF1解旋酶。提议 工作将：1）确定RECQ4亚家族酶酶和PSO2家族核酸酶如何在Fanconi贫血中起作用 独立的DNA ICL修复，确定该修复途径中涉及的其他因素，并检查疾病如何 RECQL4等位基因在机械水平上扰动其基因组维持功能； 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 这些解旋酶的生物化学上的蛋白质，并在体外重建端粒酶全酶； 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 与基因组完整性有关。为了执行这项工作，我们将采用各种经典和尖端 实验技术，从标准的体外酶试验和模型生物遗传学到接下来 生成测序，交联质谱和自定义点击化学探针的开发。 总体而言，这项工作将为了解PIF1和RECQ系列解旋酶如何提供至关重要的基本数据 协助维持基因组稳定性，并最终导致治疗靶标和治疗方法 解旋酶连接疾病。