Proteome-Wide Identification of -Methyl-2'-deoxyguanosine-Binding Proteins.

DNA is subjected to damage from various endogenous and exogenous sources of alkylating agents, resulting in alkylated DNA lesions. Among these lesions, -methyl-2'-deoxyguanosine (-Me-dG) is highly mutagenic, and it can be repaired by -alkylguanine DNA alkyltransferase and mismatch repair pathway. It, however, remains unclear whether -Me-dG in DNA can be recognized by other cellular proteins. Here, we employed a quantitative mass spectrometry-based approach to uncover reader proteins of -Me-dG in DNA when it is paired with a 2'-deoxycytidine (dC) or thymidine (dT). We were able to identify 67 and 31 candidate reader proteins for duplex DNA harboring -Me-dG:dC and -Me-dG:dT base pairs, respectively. In addition, genetic ablation of CDKN2AIP, a.k.a. CARF, one of those proteins that can recognize both the -Me-dG:dC and -Me-dG:dT base pairs, in HEK293T cells conferred augmented tolerance to -nitroso--methylurea (NMU), an alkylating agent that can induce -Me-dG in DNA. Accordingly, our LC-MS/MS quantification results revealed that the loss of CDKN2AIP led to diminished accumulation of NMU-induced -Me-dG in genomic DNA. Together, we explored the damage recognition proteins of -Me-dG using a quantitative mass spectrometry-based approach, and our results revealed an unexpected role of CDKN2AIP in sensitizing cultured cells toward a DNA methylating agent.