HMGB3 and SUB1 Bind to and Facilitate the Repair of N²-Alkylguanine Lesions in DNA

Abstract

N 2 -Alkyl-2′-deoxyguanosine (N 2 -alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The N 2 -alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for N 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 N 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 N 2 -nBu-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[a]pyrene-7,8-diol-9,10-epoxide (BPDE). SUB1 exhibits preferential binding to both the cis and trans diastereomers of N 2 -BPDE-dG over unmodified dG. On the other hand, HMGB3 binds favorably to trans-N 2 -BPDE-dG; the protein, however, does not distinguish cis-N 2 -BPDE-dG from unmodified dG. Consistently, genetic ablation of HMGB3 conferred diminished repair of trans-N 2 -BPDE-dG, but not its cis 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 N 2 -alkyl-dG lesions and documented a unique role of HMGB3 in the stereospecific recognition and repair of N 2 -BPDE-dG.