Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells

活细胞突变和突变避免的复杂机制

• 批准号: 9797176
• 负责人: Eric Alan Josephs
• 金额: $ 30.45万
• 依托单位: UNIVERSITY OF NORTH CAROLINA GREENSBORO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9797176
• 关键词: Address; Affect; Architecture; Award; Behavior; Biological Assay; Biotechnology; Cells; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; DNA; DNA Repair; DNA biosynthesis; Disease; Drug resistance; Epigenetic Process; Event; Experimental Designs; Genetic Diseases; Genetic Materials; Genome; Genomics; Goals; Health; Human; Hybrids; Laboratories; Lesion; Life; Malignant Neoplasms; Microsatellite Instability; Mismatch Repair; Molecular; Molecular Genetics; Mutation; Nature; Oligonucleotide Probes; Organism; Outcome; Pathogenicity; Pathway interactions; Play; Process; Research; Role; Sea; Source; Techniques; Toxic effect; Trees; Viral; acronyms; antimicrobial; environmental mutagens; in vivo; innovation; next generation; novel; novel therapeutics; pathogen; repaired; tumor

All organisms strive to maintain genomic fidelity in the face of agents that can damage their genetic material and the possibility that errors that can occur whenever their DNA is replicated. The ultimate goals of my research are to understand (i) how the mechanism and high-level coordination of DNA repair processes are governed by molecular, genetic, and epigenetic factors in vivo; (ii) how these factors affect diverse repair processes in different contexts to affect human health; and (iii) how clinically-important modulators of DNA repair activities and of repair-related toxicity can be leveraged as novel therapeutics. I have focused primarily on DNA mismatch repair (MMR) pathways, the pathways responsible for correcting errors that occur during DNA replication. As a primary mechanism of mutation avoidance in nearly all organisms, MMR plays a central role in many diverse processes that affect human health, from the emergence of drug resistance in infectious pathogens and cancers to the onset and treatment of somatic genetic diseases. We developed a novel assay to deconstruct the biomolecular mechanisms of MMR that uses chemically-modified oligonucleotide probes to insert targeted DNA `mismatches' directly into the genome of living cells. This assay, which we call by the acronym `SPORE,' can thus be used to directly interrogate replication-coupled repair processes like MMR quantitatively in a strand-, orientation-, and lesion-specific manner in vivo—something nearly impossible to achieve otherwise. Using the SPORE assay as a uniquely powerful baseline of approach, and in combination with next-generation biotechnologies like CRISPR and innovative experimental design, my laboratory will seek to answer the following broad-spectrum and transdisciplinary questions: · How do different molecular, genetic, and epigenetic factors affect the higher-order architecture (components and interactions), coordination, dynamics of different MMR mechanisms? How do these factors affect repair-associated toxicities? Are different molecular lesions recognized by MMR repaired according to different mechanisms and toxicities? · Do the unique repair mechanisms in pathogenic organisms represent a novel source of antimicrobial targets? · How do viral factors and environmental mutagens modulate MMR and MMR-related toxicities and by what mechanism? What is their role in hypermutation and emergence of drug resistance? · What governs the tradeoff between mutagenic and anti-mutagenic roles of MMR in microsatellite instability (MSI) diseases? · What occurs during collisions between DNA repair or other processes on DNA, and what is the nature and origin of related catastrophic mutational events? These questions are each complex in their own right and have remained difficult to answer using traditional techniques, but our unique hybrid approach provides a direct way to address each of them. The likely outcomes during the R35 award will be numerous breakthroughs in our understanding of mutational processes and how it can be manipulated in living cells; with a long-term impact being a sea-change in the ability to probe and exploit DNA damage repair mechanisms to treat disease.

所有生物体在面对可能损害其遗传物质的物质时都努力维持基因组保真度 以及复制DNA时可能发生错误的可能性是我的最终目标。 研究目的是了解 (i) DN​​A 修复过程的机制和高级协调是如何进行的 受体内分子、遗传和表观遗传因素控制；（ii）这些因素如何影响不同的修复； 不同背景下的过程如何影响人类健康；以及 (iii) DN​​A 调节剂的临床重要性 修复活性和修复相关毒性可以作为我主要关注的新疗法。 DNA错配修复（MMR）途径，负责纠正DNA错配修复过程中发生的错误的途径 DNA 复制作为几乎所有生物体避免突变的主要机制，MMR 发挥着核心作用。 从传染病耐药性的出现开始，在影响人类健康的许多不同过程中发挥着作用 我们开发了一种新的检测方法来研究病原体和癌症对体细胞遗传疾病的发病和治疗的影响。 解构 MMR 的生物分子机制，使用化学修饰的寡核苷酸探针 将“错配”的目标 DNA 直接插入活细胞的基因组中，我们将其称为“错配”。 因此，首字母缩略词“SPORE”可用于直接询问复制耦合修复过程，如 MMR 在体内以链、方向和病变特异性方式定量——这几乎是不可能的 使用 SPORE 检测作为独特且强大的基线方法，并结合起来实现其他目标。 借助 CRISPR 等下一代生物技术和创新实验设计，我的实验室将寻求 回答以下广谱和跨学科问题： · 不同的分子、遗传、 表观遗传因素影响高阶结构（成分和相互作用）、协调、 不同 MMR 机制的动态如何不同？ MMR 识别的分子损伤根据不同的机制和毒性进行修复？ 病原生物的独特修复机制代表了抗菌靶标的新来源？ 病毒因素和环境诱变剂是否会调节 MMR 和 MMR 相关毒性？ 它们在超突变和耐药性出现中的作用是什么？ MMR 在微卫星不稳定性 (MSI) 疾病中的诱变和抗诱变作用之间的权衡？ DNA 修复或 DNA 上的其他过程之间发生碰撞时会发生什么，其性质和性质是什么？ 相关灾难性突变事件的起源？这些问题本身都很复杂，并且具有以下特点： 使用传统技术仍然很难回答，但我们独特的混合方法提供了一种直接的方法 R35 颁奖期间可能出现的结果将是我们的众多突破。 了解突变过程以及如何在活细胞中操纵它并产生长期影响； 探索和利用DNA损伤修复机制来治疗疾病的能力发生了翻天覆地的变化。