NIH resubmission Deyu Li - Etheno adductome and repair pathways

项目摘要 长期以来，慢性炎症和持续感染状况与癌症风险增加有关。 越来越多的证据表明，癌症相关的炎症过程，如脂质过氧化，导致 基因组的不稳定性可能与致癌作用的发展有关。来自脂质的反应性物质 已知过氧化作用破坏DNA并形成乙烯基型加合物。以前，四种乙烯基DNA加合物 已报道的有：1，N6-乙烯基腺嘌呤（εA）、3，N4-乙烯基胞嘧啶（εC）、1，N2-乙烯基鸟嘌呤（1，N2-εG）和 N2，3-乙烯基鸟嘌呤（N2，3-εG）。这些乙烯基损伤也是由人类致癌物质的代谢产物产生的 氯乙烯。最近，发现了一种新的乙烯加合物3，N4-乙烯-5-甲基胞嘧啶（ε 5 mC）。它得到了 乙烯对5-甲基胞嘧啶的损伤，这是人类重要的表观遗传标记。到目前为止，没有关于 已报道ε 5 mC的修复和致突变性。乙烯基损伤的复制已知会导致 突变，并可能构成导致肿瘤转化的途径中的关键步骤。重要的是 在细胞中，DNA修复途径是基因组完整性的监护人，其功能是将受损的DNA恢复到其 典型状态该研究项目主要集中在两个关键的修复途径：碱基切除修复（BER）和直接修复。 逆转修复（DRR）。大多数实验，使我们目前的理解误码率和DRR是 使用DNA寡聚物进行。对于修复是如何发生的， 真核生物的DNA在染色质中紧密结合在一个复杂的DNA-蛋白质相互作用的层次结构中。在 核小体核心颗粒（NCP）是染色质组织的最基本层次，是基本的包装 由包裹在组蛋白核心周围的双链DNA组成的单位。拟议的《行动纲领》的总体目标 研究的目的是了解DNA序列背景和DNA包装到染色质中如何影响修复 乙烯内收体这个建议的中心假设是BER和DRR酶修复乙烯， 具有不同效率的损伤，并且这些独特的修复概况是1）序列背景的结果， 损伤和与酶的相互作用，以及2）染色质的蛋白质组分对修复的调节， 组蛋白在这一新假设、强有力的初步数据和创新技术的指导下， 研究了三个目标：（1）定义序列上下文效应（通过考虑5'和3'相邻 碱基）的BER和DRR酶在未包装的DNA寡聚体;（2）表征的修复概况的五个 NCP中的乙烯加合物;和（3）确定无尾和变体组蛋白蛋白提供 调节染色质修复的机制。这项研究很重要，因为它将揭示关键的 影响乙烯内收体修复的机制和关键差异，以及细胞如何最大限度地减少乙烯内收体的修复。 这些损伤的有害后果。在这项工作中获得的结果将解释在体内观察， 烷基化损伤谱，有助于我们理解突变热点和突变签名。 因此，该研究具有相当大的转化潜力，以提高我们对DNA修复的理解 研究结果可以帮助开发未来的治疗方法， 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.