The mutagenic consequences of replication-coupled DNA repair mechanisms

项目摘要 环境遗传毒素，如氧化剂、烷化剂、芳香胺、交联剂、 多环芳香族碳氢化合物和天然毒素会引起包括基础疾病在内的各种DNA损伤 位点、链间交联物和庞大的DNA碱基加合物。这些环境基因毒素在我们的 水道、食品、工业和农业化学品以及空气污染，并有可能导致 如果基因损伤得不到修复，就会发生突变和基因组不稳定。突变和基因组不稳定性可能 导致发育障碍、衰老和癌症。HMCES是一种复制偶联修复蛋白， 对单链DNA基本位点作出反应，并防止它们被AP-内切酶切割。基本站点有 一种常见的由环境遗传毒素引起的病变。我的初步结果表明，HMCES可以预防 基因组不稳定和突变，我假设它促进了一种更忠实的修复机制 例如模板切换或分叉反转。对于这项提案的K99阶段，我将学习执行简短和 长期诱变试验和DNA深度测序方法详细了解HMCES 防止人类细胞中的突变和基因组不稳定，并促进更多的无错误修复。这项工作将 为表征链特异性复制压力的研究(R00)创建技术基础和蓝图 反应以及链特异性障碍和环境基因毒素如何导致领先和滞后 链突变。在领先链和滞后链上复制之间存在核心差异。脱氧核糖核酸 复制在前导链上连续进行，在滞后链上不连续地通过一系列 对事件的谴责。我假设链特异的损伤和障碍会产生差异复制 应激反应，并以不同方式增强诱变作用。我将描述滞后和领先的链条压力 使用不偏不倚的方法作出回应。此外，我将确定特定于链的应力和 基于深度测序的基因毒素对致突变链偏向的研究。归根结底，这 提案将推进环境毒理学和DNA修复领域，导致范式转变 发现。

Project Summary Environmental genotoxins such as oxidation agents, alkylating agents, aromatic amines, crosslinking agents, polycyclic aromatic hydrocarbons, and natural toxins induce a full spectrum of DNA lesions including abasic sites, interstrand crosslinks, and bulky DNA base adducts. These environmental genotoxins are found in our waterways, food, industrial and agricultural chemicals, and air pollution and have the potential to induce mutagenesis and genomic instability if genetic lesions are not repaired. Mutagenesis and genomic instability can lead to developmental disorders, aging, and cancers. HMCES is a replication-coupled repair protein that responds to single-strand DNA abasic sites and prevents their cleavage by AP-endonucleases. Abasic sites are a common lesion caused by environmental genotoxins. My preliminary results suggest that HMCES prevents both genomic instability and mutagenesis, and I hypothesize that it promotes a more faithful repair mechanism such as template switching or fork reversal. For the K99-phase of this proposal I will learn to perform short and long-term mutagenesis assays and DNA deep sequencing methods to understand in detail how HMCES prevents mutagenesis and genomic instability in human cells and promotes more error-free repair. This work will create a technical foundation and blueprint for studies (R00) characterizing the strand-specific replication stress response and how strand-specific obstacles and environmental genotoxins contribute to leading and lagging strand mutagenesis. There are core differences between replication on the leading and lagging strands. DNA replication occurs continuously on the leading strand and discontinuously on the lagging strand through a series of repriming events. I hypothesize that strand-specific lesions and obstacles generate a differential replication stress response, and potentiate mutagenesis differently. I will characterize the lagging and leading strand stress responses using unbiased approaches. Further, I will determine the consequences of strand-specific stress and genotoxins on mutagenic strand bias using deep sequencing-based mutagenesis assays. Ultimately, this proposal will advance the environmental toxicology and DNA repair fields leading to paradigm shifting discoveries.