HMGB3 and SUB1 Bind to and Facilitate the Repair of <i>N</i>²-Alkylguanine Lesions in DNA

<i>N</i>²-Alkyl-2'-deoxyguanosine (<i>N</i>²-alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The <i>N</i>²-alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for <i>N</i>²-alkyl-dG lesions remain incompletely elucidated. By utilizing a photo-cross-linking coupled with mass spectrometry-based quantitative proteomic analysis, we identified a series of candidate <i>N</i>²-alkyl-dG-binding proteins. We found that two of these proteins, i.e., high-mobility group protein B3 (HMGB3) and SUB1, could bind directly to <i>N</i>²-<i>n</i>Bu-dG-containing duplex DNA in vitro and promote the repair of this lesion in cultured human cells. In addition, HMGB3 and SUB1 protected cells against benzo[<i>a</i>]pyrene-7,8-diol-9,10-epoxide (BPDE). SUB1 exhibits preferential binding to both the cis and trans diastereomers of <i>N</i>²-BPDE-dG over unmodified dG. On the other hand, HMGB3 binds favorably to <i>trans</i>-<i>N</i>²-BPDE-dG; the protein, however, does not distinguish <i>cis</i>-<i>N</i>²-BPDE-dG from unmodified dG. Consistently, genetic ablation of <i>HMGB3</i> conferred diminished repair of <i>trans</i>-<i>N</i>²-BPDE-dG, but not its <i>cis</i> counterpart, whereas loss of SUB1 conferred attenuated repair of both diastereomers. Together, we identified proteins involved in the cellular sensing and repair of minor-groove <i>N</i>²-alkyl-dG lesions and documented a unique role of HMGB3 in the stereospecific recognition and repair of <i>N</i>²-BPDE-dG.