Identification and characterizations of DNA N2-alkylguanine-binding proteins

DNA alkylation at the N2 position of guanine (N2-dG) is a prevalent type of DNA minor-groove lesions arising from various exogenous environmental contaminants and endogenous cellular processes. These N2-alkyl-dG lesions can induce G → T mutations during transcription if left unrepaired. However, the repair pathways of N2-alkyl-dG lesions remain incompletely elucidated. In this study, we identified a series of potential N2-alkyl-dG-binding proteins utilizing an affinity pulldown coupled with quantitative proteomic approach. We investigated their roles in DNA damage response and repair of these lesions. High-mobility group protein B3 (HMGB3) and activated RNA polymerase II transcriptional coactivator P15 (SUB1) exhibited preferential binding toward N2-nBudG-containing duplex DNA in quantitative proteomics analysis and in vitro binding assay using recombinant proteins. The presence of HMGB3 and SUB1 protected cells against alkylating agents (e.g., BPDE). Both HMGB3 and SUB1 modulated the repair of N2-nBudG and trans-N2-BPDE-dG in genomic DNA, while HMGB3 wasn’t involved in the repair of cis-N2-BPDE-dG. Together, our findings provided new knowledge about the cellular sensing and repair of minor-groove N2-alkyl-dG lesions.