Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA

Abstract

Methylglyoxal, a known endogenous and environmental mutagen, is a reactive alpha-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a 'primed template' DNA and the exonuclease-deficient Klenow fragment (KF(exo-)) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS-PAGE, we found that methylglyoxal induced a DNA-KF(exo-) crosslink. The specific binding complex of KF(exo-) and 'primed template' DNA was necessary for formation of the DNA-KF(exo-) crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2'-deoxyguanosine was incubated with Nalpha-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA-DNA polymerase crosslink.

Figure 1

Formation of a DNA–KF^exo– crosslink induced by methylglyoxal. The mixture of DNA (T-1/P-1) and KF^exo– was incubated with H₂O (lane 1), 10 mM glyoxal (lane 2) or 10 mM methylglyoxal (lane 3) at 37°C for 60 min, as described in Materials and Methods. Lane 4 was identical to lane 3, except that the DNA was absent. Lanes 5 and 6 were identical to lanes 2 and 3, respectively, except that the incubation was for 120 min.

Figure 2

Time course of DNA–KF^exo– crosslink formation by methylglyoxal. T-1/P-1 and KF^exo– were incubated with 10 mM methylglyoxal, as described in Materials and Methods, for the indicated time periods.

Figure 3

Influence of DNA sequence upon the methylglyoxal-induced DNA–KF^exo– crosslink. The ‘primed template’ and KF^exo– were incubated with the indicated concentrations of methylglyoxal (MG) at 37°C for 60 min.

Figure 4

Influence of NaBH₄ reduction of the methylglyoxal-induced DNA–KF^exo– crosslink. T-1/P-1 and KF^exo– were incubated with 10 mM methylglyoxal at 37°C for the indicated period (1st incubation) and were additionally incubated for 60 min in the presence or absence of NaBH₄ (2nd incubation).

Figure 5

Methylglyoxal crosslinks dG and AcLys. (A) dG and AcLys were incubated in the absence of methylglyoxal. (B) AcLys, (C) dG and (D) dG and AcLys were incubated with 20 mM methylglyoxal at 37°C for 10 days. The reaction mixture was analyzed by anion exchange HPLC.

Figure 6

Time course of dG–AcLys (closed circles) and dG–AcCys (open circles) crosslink formation by methylglyoxal. dG and AcLys/AcCys were incubated with 20 mM methylglyoxal as described in Materials and Methods for the indicated time periods.