Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells

所有生物体在面对可能破坏其遗传物质的物质时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。我的终极目标 研究的目的是了解（i）DNA修复过程的机制和高级协调是如何 受体内分子、遗传和表观遗传因素的影响;（ii）这些因素如何影响不同的修复 过程在不同的情况下，以影响人类健康;和（iii）如何临床上重要的调节剂的DNA 修复活性和修复相关毒性的降低可以作为新的治疗剂。我主要关注 DNA错配修复（MMR）途径，负责纠正错误的途径， DNA复制。作为几乎所有生物体避免突变的主要机制，MMR在基因突变中起着核心作用。 在影响人类健康的许多不同过程中发挥作用，从感染性疾病的耐药性出现， 病原体和癌症的发病和治疗的躯体遗传疾病。我们开发了一种新的检测方法 解构MMR的生物分子机制，该机制使用化学修饰的寡核苷酸探针， 将靶向DNA“错配”直接插入活细胞的基因组中。我们称之为 因此，可以使用缩写为“SPORE”直接询问复制偶联修复过程，如MMR 在体内以链、方向和病变特异性的方式定量地检测-这几乎是不可能的， 实现否则。使用SPORE检测试剂盒作为唯一强大的基线方法，并结合 有了CRISPR等新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，遗传， 和表观遗传因素影响高阶结构（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毒性？是不同 根据不同的机制和毒性修复MMR识别的分子损伤？·做 病原微生物中独特的修复机制代表了抗菌靶标的新来源？·如何 病毒因素和环境诱变剂是否调节MMR和MMR相关毒性，以及通过什么调节 机制？它们在超突变和耐药性出现中的作用是什么？·什么支配着 MMR在微卫星不稳定性（MSI）疾病中的致突变和抗突变作用之间的权衡？· DNA修复或DNA上的其他过程之间的碰撞过程中会发生什么，其性质是什么， 相关灾难性突变事件的起源这些问题本身就很复杂， 使用传统技术仍然难以回答，但我们独特的混合方法提供了一种直接的方法， 来解决每一个问题。在R35奖项期间可能的结果将是我们在技术上的许多突破。 了解突变过程以及如何在活细胞中操纵它;具有长期影响 这是探测和利用DNA损伤修复机制治疗疾病能力的巨大变化。

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.