Molecular Mechanisms Of Modular Nuclease Domains

项目摘要/摘要 核酸酶是一类能在多种细胞过程中水解核酸底物的酶。 它们的活性是维持DNA复制、修复和基因组完整性所必需的 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调控失调有关。包括林奇综合征，一种 与结直肠癌和子宫内膜癌、亨廷顿氏病以及 不孕不育。细菌特有的核酸酶也是抗生素的潜在靶标，并完全理解 核酸酶生物化学为发现新的药物靶点和开发新的核酸酶铺平了道路 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。 我们将使用DNA修复过程中的蛋白质来模拟核酸酶活性，并确定调控和 具体步骤。使用容易处理的DNA错配修复途径来拼写检查新复制的DNA，我们 将确定如何赋予固有的非特定核酸酶特异性。在这个过程中的蛋白质已经被 参与减数分裂重组，并在调节三核苷酸重复扩增中发挥作用 这表明相关的核酸酶活性是模块化的。这项工作阐述了这种增持的起源和 提供了所有这些通路如何将底物专一性传递给 核裂解部位。在细菌中，同源重组是获得抗生素耐药性的一种方法。MutS2， 一种错配识别复合体的同系物，已被认为与几种细菌有关 路径。它的作用机制以及它是否遵循规范错配修复所建立的范式 蛋白质是不清楚的，通过这里描述的机械性工作来解决。MutS2的核酸酶结构域 在整个生物学中被发现是具有不同特性和功能的蛋白质的融合。我们将确定 这个核酸酶结构域的模块化，它如何通过其他结构域实现特异性，并测试其潜在的 适应作为一种基因编辑工具。我们还将调查新的病毒的特异性和调控机制 在古生菌中发现了一种多功能的核蛋白，它参与了多种DNA修复过程 以及分枝杆菌，包括引起结核病的结核分枝杆菌。这将提供 在DNA修复过程中利用核素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支持，这些模型最终可以用于检测 并开发治疗人类疾病的疗法。此外，这项工作提供了对核酸酶如何 都是受调控的，并将指导未来对其他细胞途径的研究。

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.