DNA is susceptible to damage from alkylating agents resulting from endogenous metabolism and exogenous exposure, where alkylation can occur in the major- and minor-groove of DNA. The presence of alkylation adducts on DNA can block DNA both replication, and transcription, and induce mutations in these processes. The ubiquitous nature of alkylating agents and their detrimental effects require cells to have mechanisms to sense and repair alkylated DNA lesions. However, there is a dearth of information about how cells sense the presence of alkylation in the major- and minor-grooves of the DNA double helix. Furthermore, cells harness alkylation as a mechanism for regulating gene expression; hence, questions remain as to how cells differentiate between these DNA epigenetic marks and alkylation DNA damage. Here, we employed an unbiased quantitative proteomic approach to explore how cells recognize major- and minor-groove DNA alkylation products. We prepared oligonucleotides with alkylation located either in the major- (N6 position of adenine or N4 position of cytosine) or minor-groove (N2 position of guanine) of DNA. Additional oligonucleotides were synthesized containing either a site-specific DNA epigenetic marker (N6-Me-dA) or site-specific DNA damage products (N6-n-butyl-dA or O6-Me-dG). The synthesized site-specific oligonucleotides were incubated with nuclear lysate obtained from cells cultured in light- and heavy stable isotope-labeled (SILAC) media for the detection of readers of alkylated DNA bases. We were able to identify a number of putative reader proteins for these alkylated nucleosides in duplex DNA. Future work includes validation of these interactions with the use of purified recombinant proteins and characterizations of the biological functions of these interactions.

Identification and functional characterizations of recognition proteins for major- and minor-groove DNA damage

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