Molecular mechanisms of modular nuclease domains

模块化核酸酶结构域的分子机制

• 批准号: 10274492
• 负责人: Carol M Manhart
• 金额: $ 34.96万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10274492
• 关键词: Address; Antibiotic Resistance; Antibiotics; Archaea; Area; Bacteria; Biochemistry; Biological Process; Biology; Biotechnology; Cell physiology; Colorectal Cancer; Complex; DNA; DNA Repair; DNA biosynthesis; Development; Disease; Disease model; Endometrial Carcinoma; Enzymes; Failure; Future; Genes; Genetic Recombination; Genus Mycobacterium; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Huntington Disease; Infertility; Link; Maintenance; Meiotic Recombination; Methods; Mismatch Repair; Modeling; Molecular; Mycobacterium tuberculosis; Nucleic Acids; Organism; Pathway interactions; Play; Process; Proteins; Regulation; Research; Role; Site; Specificity; Substrate Specificity; Syndrome; Testing; Therapeutic; Therapeutic Agents; Trinucleotide Repeat Expansion; Tuberculosis; Work; cancer predisposition; genome integrity; homologous recombination; human disease; insight; interest; new therapeutic target; novel; nuclease; programs; repaired; tool

Project Summary/Abstract Nucleases are a class of enzyme that hydrolyze nucleic acid substrate in a variety of cellular processes. Their activity is a requirement for the maintenance of genomic integrity in DNA replication, repair, and recombination. Understanding nuclease regulation and specificity in these processes is critical for modeling these fundamental pathways and human diseases linked to dysregulation. This includes Lynch syndrome, a cancer predisposition syndrome linked to colorectal and endometrial cancers, Huntington’s disease, and infertility. Nucleases unique to bacteria are also potential targets for antibiotics and a complete understanding of nuclease biochemistry paves the way for the discovery of new drug targets and exploiting nucleases as new biotechnological tools and therapeutic agents. Characterizing and developing novel nucleases is a future area of interest for my research program. We will use proteins in DNA repair processes to model nuclease activity and determine regulation and specificity steps. Using the tractable DNA mismatch repair pathway which spellchecks newly replicated DNA, we will identify how inherently nonspecific nucleases can be given specificity. Proteins in this process have been co-opted for meiotic recombination and also play a role in the regulation of trinucleotide repeat expansions indicating that the associated nuclease activity is modular. This work addresses the origins of this co-option and provides missing mechanistic detail for how all of these pathways communicate substrate specificity to nucleolytic sites. In bacteria, homologous recombination is a method for acquiring antibiotic resistance. MutS2, a homolog to mismatch recognition complexes, has been implicated in several bacteria as being involved in this pathway. Its mechanisms of action and whether it follows paradigms established by canonical mismatch repair proteins are not clear and are addressed by mechanistic work described here. The nuclease domain of MutS2 is found throughout biology as a fusion to proteins with diverse specificities and functions. We will determine the modularity of this nuclease domain, how it achieves specificity by other domains, and test its potential for adaptation as a gene editing tool. We will also investigate the specificity and regulatory mechanisms of the newly discovered NucS protein which is multi-functional, and is implicated in multiple DNA repair processes in archaea and mycobacteria, including Mycobacterium tuberculosis, the bacteria that causes tuberculosis. This will provide key evidence for adaptation processes of organisms that utilize NucS in DNA repair processes. Our work will provide an underpinning for complex mechanistic models that can be ultimately used to detect and develop therapeutics for human disease. In addition, this work provides general insight into how nucleases are regulated and will guide future studies in other cellular pathways.

项目摘要/摘要 核酸酶是在各种细胞过程中水解核酸底物的一类酶。 它们的活性是在DNA复制，维修和 重组。了解这些过程中的核酸酶调节和特异性对于建模至关重要 这些基本途径和人类疾病与失调有关。这包括林奇综合症，一个 癌症易感综合征与结直肠癌和子宫内膜癌，亨廷顿氏病以及 不育。细菌独有的核酸酶也是抗生素的潜在靶标，并且对 核酸酶生物化学为发现新药物靶标的铺平了道路，并利用核质量作为新的 生物技术工具和治疗剂。表征和发展新颖的核武器是未来的领域 我的研究计划感兴趣。 我们将在DNA修复过程中使用蛋白质来建模核酸酶活性并确定调节和 特异性步骤。使用可拖动的DNA不匹配修复途径，该途径拼写了新复制的DNA，我们 将确定如何固有的非特异性核能给予特异性。在此过程中的蛋白质已经 选择减数分裂重组，并在调节三核苷酸重复扩张中发挥作用 表明相关的核酸酶活性是模块化的。这项工作涉及此选择的起源和 为所有这些途径如何将基板特异性传达给 核解位点。在细菌中，同源重组是一种获得抗生素耐药性的方法。 muts2， 在几种细菌中暗示了不匹配识别复合物的同源物 路径。它的作用机理及其是否遵循规范不匹配修复确定的范例 蛋白质尚不清楚，并通过此处描述的机械工作来解决。 muts2的核酸酶结构域 在整个生物学中都被发现是与具有潜水员规格和功能的蛋白质融合。我们将确定 该核酸酶结构域的模块化，如何通过其他域达到特异性，并测试其潜力 作为基因编辑工具的适应。我们还将研究新近的特异性和调节机制 发现的NUC蛋白是多功能的，与古细菌的多个DNA修复过程有关 分枝杆菌，包括结核分枝杆菌，导致结核病的细菌。这将提供 在DNA修复过程中利用NUC的生物体适应过程的主要证据。 我们的工作将为复杂的机械模型提供基础，最终可用于检测 并开发人类疾病的疗法。此外，这项工作提供了有关核能如何的一般见解 受调节，并将指导其他细胞途径的未来研究。