NIH resubmission Deyu Li - Etheno adductome and repair pathways

项目总结 长期以来，慢性炎症和持续感染状况一直与癌症风险增加有关。 越来越多的证据表明，与癌症相关的炎症过程，如脂质过氧化，会导致 基因组的不稳定性可能与癌症的发生发展有关。脂类中的活性物质 已知过氧化会损伤DNA并形成亚乙烯型加合物。此前，四种亚甲基DNA加合物 已报道：1，N6-乙烯腺嘌呤(εA)，3，N4-乙胞嘧啶(εC)，1，N-乙基鸟嘌呤(1，N-εG)，和 N，3-亚乙基鸟嘌呤(N2，3-εG)。这些乙烯损伤也是由人类致癌物质的代谢物产生的。 氯乙烯。最近，一个新的亚乙基加合物3，N4-亚乙基-5-甲基胞嘧啶(ε5mC)被鉴定出来。它承载着 对人类重要的表观遗传标记5-甲基胞嘧啶的乙烯损伤。到目前为止，还没有关于 ε5mC的修复和致突变性已有报道。已知乙烯基损伤的复制会导致 突变，可能是导致肿瘤转化的关键一步。重要的是 细胞，DNA修复途径是基因组完整性和功能的守护者，将受损的DNA恢复到其 正则状态。本研究主要围绕碱基切除修复和直接修复两条关键修复途径展开 反向修复(DRR)。引起我们目前对误码率和DRR理解的大多数实验是 使用DNA寡聚体进行。在修复如何在上下文中发生的知识方面存在着根本的差距 在染色质中，真核DNA在DNA-蛋白质相互作用的复杂层次中被压缩。在 染色质组织的最基本层面，是核小体核心颗粒(NCP)的基本包装 由包裹在组蛋白核心上的ds-DNA组成的单位。建议的总体目标是 研究是为了了解DNA序列背景和DNA进入染色质的包装如何影响修复 亚烯基内收体的。这一提议的中心假设是BER和DRR酶修复乙烯 不同效率的损伤，这些独特的修复特征是1)序列背景的结果 损伤和与酶的相互作用以及2)染色质的蛋白质成分对修复的调节， 组蛋白。在这一新的假设、强大的初步数据和创新技术的指导下，该提案 研究的三个目的是：(1)定义序列语境效应(通过考虑5‘和3’邻近的 碱基)的BER和DRR酶在未包装的DNA寡聚体中；(2)表征了五种酶的修复情况 NCP中的乙烯加合物；以及(3)确定无尾和变异组蛋白在多大程度上提供 染色质的调制修复机制。这项拟议的研究意义重大，因为它将揭示关键 影响内收体修复的机制和关键差异，以及细胞如何最小化 这些损伤的有害后果。这项工作中获得的结果将解释在体内观察到的 烷基化损伤谱并有助于我们理解突变热点和突变特征。 因此，这项研究对增进我们对DNA修复的理解具有相当大的翻译潜力 这一结果有助于开发未来提高细胞防御能力的治疗方法。 对抗基因组的不稳定性。

PROJECT SUMMARY Chronic inflammation and persistent infection conditions have long been associated with increased risk of cancer. Growing evidence suggests that cancer-associated inflammatory processes, such as lipid peroxidation, cause genomic instability that can be linked to the development of carcinogenesis. Reactive species from lipid peroxidation are known to damage DNA and form etheno-type adducts. Previously, four etheno DNA adducts have been reported: 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC), 1,N2-ethenoguanine (1,N2-εG), and N2,3-ethenoguanine (N2,3-εG). These etheno lesions are also generated by metabolites of the human carcinogen vinyl chloride. Recently, a new etheno adduct, 3,N4-etheno-5-methylcytosine (ε5mC), was identified. It bears the etheno damage on 5-methylcytosine, an important epigenetic marker in humans. Thus far, no information on the repair and mutagenicity of ε5mC has been reported. Replication of the etheno lesions is known to cause mutations and may constitute a critical step in the pathway leading to neoplastic transformation. Importantly for cells, DNA repair pathways are the guardians of genomic integrity and function to return damaged DNA to its canonical state. This research project focuses on two key repair pathways: base excision repair (BER) and direct reversal repair (DRR). Most of the experiments that give rise to our current understanding of BER and DRR were conducted using DNA oligomers. There is a fundamental gap in knowledge of how repair occurs in the context of chromatin, where eukaryotic DNA is compacted in a complex hierarchy of DNA-protein interactions. At the most fundamental level of chromatin organization, the nucleosome core particle (NCP) is the basic packaging unit that is comprised of ds-DNA wrapped around a histone protein core. The overarching goal of the proposed research is to understand how DNA sequence context and the packaging of DNA into chromatin influence repair of the etheno adductome. The central hypothesis of this proposal is that BER and DRR enzymes repair etheno lesions with different efficiencies, and these distinctive repair profiles are the result of 1) sequence context of the lesion and interactions with the enzyme and 2) modulation of repair by the protein component of chromatin, the histones. Guided by this novel hypothesis, strong preliminary data, and innovative techniques, the proposal investigates three aims that: (1) define the sequence context effects (by considering the 5’ and 3’ neighboring bases) of BER and DRR enzymes in unpackaged DNA oligomers; (2) characterize the repair profiles of the five etheno adducts in NCPs; and (3) determine the extent to which tailless and variant histone proteins provide a mechanism of modulating repair in chromatin. The proposed research is significant because it will reveal key mechanisms and critical differences that influence repair of the etheno adductome and how cells minimize the harmful consequences of these lesions. The results obtained in this work will explain in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots and mutational signatures. Therefore, the research has considerable translational potential to enhance our understanding of DNA repair and the results can assist in the development of future therapeutic treatments that improve cellular defenses against genomic instability.