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.

项目总结/摘要 核酸酶是一类在多种细胞过程中水解核酸底物的酶。 它们的活性是在DNA复制、修复和转录中维持基因组完整性的必要条件。 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。这包括林奇综合征， 与结直肠癌和子宫内膜癌、亨廷顿病有关的癌症易感综合征，以及 不孕细菌特有的核酸酶也是抗生素的潜在靶点， 核酸酶生物化学为发现新的药物靶点和开发核酸酶作为新的药物靶点铺平了道路。 生物技术工具和治疗剂。表征和开发新型核酸酶是一个未来的领域 对我的研究项目感兴趣 我们将在DNA修复过程中使用蛋白质来模拟核酸酶活性，并确定调控和 具体步骤。使用易处理的DNA错配修复途径，拼写检查新复制的DNA，我们 将确定如何赋予固有非特异性核酸酶特异性。蛋白质在这个过程中， 共同选择减数分裂重组，并在三核苷酸重复扩增的调节中发挥作用 表明相关的核酸酶活性是模块化的。这项工作解决了这种共同选择的起源， 提供了所有这些途径如何传递底物特异性的缺失机制细节， 溶核位点在细菌中，同源重组是获得抗生素抗性的方法。MutS2， 错配识别复合物的同源物，已经在几种细菌中被牵连，因为它们参与了这一过程。 通路它的作用机制以及它是否遵循典型错配修复建立的范例 蛋白质是不清楚的，并且通过这里描述的机械工作来解决。MutS 2的核酸酶结构域 在整个生物学中被发现是具有不同特异性和功能的蛋白质的融合体。康贝特人将以 这种核酸酶结构域的模块性，它如何通过其他结构域实现特异性，并测试其对 适应作为基因编辑工具。我们还将调查新的特异性和调节机制， 发现了多功能的NucS蛋白，并涉及古细菌中的多种DNA修复过程 和分枝杆菌，包括结核分枝杆菌，引起结核病的细菌。这将提供 在DNA修复过程中利用NucS的生物体适应过程的关键证据。 我们的工作将为复杂的机械模型提供基础，这些模型最终可用于检测 and develop发展therapeutic疗法for human人类disease疾病.此外，这项工作提供了一般的洞察如何核酸酶 是受调控的，并将指导其他细胞途径的未来研究。