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.

所有生物都在努力维持可能损害其遗传物质的药物的基因组忠诚度 以及每当复制其DNA时可能出现的错误的可能性。我的最终目标 研究将了解（i）DNA修复过程的机制和高级协调如何 由体内的分子，遗传和表观遗传因子约束； （ii）这些因素如何影响潜水员的维修 在不同情况下影响人类健康的过程； （iii）DNA的临床重要调节剂如何 维修活动和与修复有关的毒性可以用作新的疗法。我专注于小学 在DNA不匹配修复（MMR）途径上，负责纠正错误的途径 DNA复制。作为几乎所有生物中突变避免突变的主要机制，MMR扮演着中央 在影响人类健康的许多潜水过程中的作用，从传染性的耐药性出现 病原体和癌症开始和治疗体细胞遗传疾病。我们开发了一种新颖的测定法 解构使用化学改性寡核苷酸问题的MMR的生物分子机制 将靶向的DNA“不匹配”直接插入活细胞的基因组中。这个测定法，我们通过 因此，可以使用首字母缩写“孢子”来直接询问复制耦合修复过程（例如MMR） 以链，定向和病变特定的方式进行定量 - 几乎不可能 否则就可以实现。将孢子测定作为一种独特的方法基线，并组合 凭借CRISPR和创新实验设计等下一代生物技术，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，如何 和表观遗传因素会影响高阶架构（组成和相互作用），协调性，， 不同MMR机制的动力学？这些因素如何影响维修相关的毒性？是不同的 根据不同机制和毒性修复的MMR识别的分子病变？ ·做 致病生物中的独特修复机制代表了抗菌靶标的新颖来源？ · 如何 病毒因素和环境诱变剂调节MMR和与MMR相关的毒性，以及什么 机制？他们在耐药性的超名和出现中的作用是什么？ ·是什么控制 MMR在微卫星不稳定性（MSI）疾病中的诱变和抗杀菌作用之间的权衡？ · DNA修复或DNA上其他过程之间发生碰撞时发生的情况，什么是性质和 相关灾难性突变事件的起源？这些问题本身都是复杂的，并且 使用传统技术仍然很难回答，但是我们独特的混合方法提供了一种直接的方式 解决每个人。 R35奖中的可能结果将是我们的许多突破 了解突变过程及其在活细胞中如何操纵的过程；长期影响 作为探测和利用DNA损伤修复机制治疗疾病的能力的海洋变化。