Abstract
O(2)- and O(4)-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a quantitative proteomic method to discover regioisomeric O(2)- and O(4)-n-butylthymidine (O(2)- and O(4)-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O(2)-nBudT- and O(4)-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O(2)-nBudT-containing DNA, whereas three high-mobility group (HMG) proteins (i.e., HMGB1, HMGB2, and mitochondrial transcription factor A (TFAM)) exhibit preferential binding to O(4)-nBudT-bearing DNA. We further demonstrated that TFAM binds directly and selectively with O(4)-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O(4)-nBudT and O(4)-pyridyloxobutylthymidine, which is a DNA adduct induced by tobacco-specific N-nitrosamines, in vitro and in human cells. Together, we explored, for the first time, the interaction proteomes of O-alkyldT lesions, and our study expanded the functions of TFAM by revealing its capability in the recognition of O(4)-alkyldT-bearing DNA and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.