Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells

面对可能破坏其遗传物质的病原体，所有有机体都努力保持基因组的保真度。 以及当他们的DNA被复制时可能发生的错误。我的最终目标是 研究的目的是了解(I)DNA修复过程的机制和高层协调是如何 体内受分子、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 不同背景下影响人类健康的过程；以及(Iii)DNA的临床重要调节物 修复活动和与修复相关的毒性可作为新的治疗药物加以利用。我主要专注于 在DNA错配修复(MMR)通路中，负责纠正在 DNA复制。作为几乎所有生物避免突变的主要机制，MMR发挥着核心作用。 在影响人类健康的许多不同过程中发挥作用，从出现耐药性到感染性 病原体和癌症与躯体遗传病的发病和治疗有关。我们开发了一种新的分析方法 解构MMR的生物分子机制，使用化学修饰的寡核苷酸探针来 将靶DNA“错配”直接插入活细胞的基因组中。这种化验，我们称之为 因此，首字母缩写‘SPORE’可用于直接询问复制耦合修复过程，如MMR 在体内以链、定向和损伤特异的方式定量--这几乎是不可能的 取得不同的成就。使用孢子分析作为唯一强大的基线方法，并结合 借助CRISPR等下一代生物技术和创新的实验设计，我的实验室将寻求 回答以下广谱和跨学科的问题：·不同的分子、基因、 表观遗传因素影响更高阶的架构(组件和相互作用)、协调、 不同MMR机制的动力学？这些因素如何影响修复相关的毒性？是不同的 MMR识别的分子损伤是否根据不同的机制和毒性进行修复？ 病原生物体中独特的修复机制代表了抗菌靶标的新来源？ 病毒因子和环境诱变剂是否调节MMR和MMR相关的毒性以及通过什么调节 机械装置？它们在超突变和耐药性产生中起什么作用？·是什么支配着 微卫星不稳定(MSI)疾病中MMR的致突变和抗突变作用之间的权衡？ 在DNA修复或DNA上的其他过程之间发生碰撞时会发生什么，性质和 相关灾难性突变事件的起源？这些问题各自都很复杂，都有 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 来解决他们中的每一个人。在R35颁奖期间，可能的结果将是我们的 了解突变过程及其如何在活细胞中被操纵；具有长期影响 探测和利用DNA损伤修复机制治疗疾病的能力发生了翻天覆地的变化。

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.