Efficient and accurate bypass of N2-(1-carboxyethyl)-2'-deoxyguanosine by DinB DNA polymerase 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 N(2)-dG 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 N(2)-CEdG could be detected at a frequency of one lesion per 10(7) 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 kappa), opposite the N(2)-CEdG is both accurate and efficient. Taken together, our data support that N(2)-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.
Yuan
Bifeng
B
Department of Chemistry and Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403, USA.
Cao
Huachuan
H
Jiang
Yong
Y
Hong
Haizheng
H
Wang
Yinsheng
Y
D001483:Base Sequence; D018736:DNA Adducts / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011765:Pyruvaldehyde / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000099270:Y-Family DNA Polymerases
D018736:DNA Adducts; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D011765:Pyruvaldehyde; C400382:N(2)-(1-carboxyethyl)deoxyguanosine; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases