NIH resubmission Deyu Li - Etheno adductome and repair pathways

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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.