Cellular DNA is constantly assaulted by several types of reactive oxygen species (ROS). These ROS insults can result in various DNA modifications ranging from small lesions like base modifications to large bulky lesions like DNA-Protein crosslinks (DPCs). It is important to identify these DNA damage endpoints since each has a unique fate in DNA damage repair and a specific effect on cell survival. Small lesions can easily bypass through the repair mechanism and lead to specific types of point mutations while the bulkier lesions can interfere with DNA replication, and if left unrepaired they can lead to cell death. Among DNA nuclear bases guanine (1.3 V vs NHE) has the lowest oxidation potential, and is readily oxidized by ROS. 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8OdG) is the most abundant primary oxidation product of 2'-deoxyguanosine (dG). 8OdG has been found to have an even lower oxidation potential (0.7 V vs NHE) than dG resulting to its facile oxidation, forming several hyperoxidation lesions including spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). However these hyperoxidation lesions have limited detection in-vivo. We hypothesize that due to the high abundance of surrounding protein's readily oxidizable amino acid side chains and relative low abundance of 8OdG, hyperoxidative lesion formation is limited in cells. This leads to the observed 8OdG and formation of DPCs. An in-vitro mutagenesis study was done to test the hypothesis. A 392mer DNA was treated with singlet oxygen generator, Rose Bengal with and without protein. Loss of dG was observed in the presence of singlet oxygen and presence of protein enhances dA mis-incorporation opposite to dG suggesting accumulation of 8OdG in the presence of proteins. When HeLa cells were treated with singlet oxygen generator in a concentration dependent manner we observed an increase in DPCs that correlates with HeLa cell death.

Influence of proteins in DNA damage: DNA-protein crosslink formation and lesion changes

248th National Meeting (2014)

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