Efficient and accurate bypass of N2-(1-carboxyethyl)-2′-deoxyguanosine by DinB DNA polyrnerase in vitro and in vivo

DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of WAG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N-2-(1-carboxyethyl)-2'-deoxyguanosine (N-2-CEdG) as the major stable DNA adduct. Here, we found that N2-CEdG could be detected at a frequency of one lesion per 107 nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N-2-CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N-2-CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N-2-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase K), opposite the N-2-CEdG is both accurate and efficient. Taken together, our data support that N2-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.