Repair of oxidative damage in DNA induced by photoactivated methylene blue in human lymphoblastoid whole cell extracts
Oxidative damage on DNA is an incessant, inevitable result of cellular metabolic activity as well as environmental carcinogens. Oxidative damage produces many different lesions. A highly specific substrate containing mainly 8-hydroxyguanine lesion was generated and an in vitro DNA repair synthesis assay was developed using human lymphoblastoid whole cell extracts. Photoactivated methylene blue introduced 1.58 Formamidopyrimidine glycosylase sensitive sites into the plasmid. The damage specific repair incorporation of deoxycytidine monophosphate by normal whole cell extracts was 10 times more efficient per 8-Hydroxyguanine than per (6-4) photoproduct. The rapid rate of nicking by normal whole cell extracts implies that the lesion is recognized efficiently and that damage specific nicking was not the rate limiting step in the repair of 8-Hydroxyguanine. Based on the difference in rate and amount of nucleotide specific incorporation, more than one pathway was indicated for repair of this oxidative lesion. A model, proposing single base replacement and small patch (6 nucleotides) size repair was designed. The fraction of nucleotides processed by normal cell extracts through single base replacement is 0.38 and the fraction of nucleotides processed through the small patch repair was 0.62. Repair of 8-Hydroxyguanine by normal whole cell extracts occurred predominantly through small patch repair.