3,N4-ethenocytosine, a highly mutagenic adduct, is a primary substrate for Escherichia coli double-stranded uracil-DNA glycosylase and human mismatch-specific thymine-DNA glycosylase

Abstract

Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3,N4-ethenocytosine (epsilonC), 1, N6-ethenoadenine (epsilonA), N2,3-ethenoguanine, and 1, N2-ethenoguanine cause mutations. The epsilonA residues are excised by the human and the Escherichia coli 3-methyladenine-DNA glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repairing epsilonC residues have not yet been described. We have identified two homologous proteins present in human cells and E. coli that remove epsilonC residues by a DNA glycosylase activity. The human enzyme is an activity of the mismatch-specific thymine-DNA glycosylase (hTDG). The bacterial enzyme is the double-stranded uracil-DNA glycosylase (dsUDG) that is the homologue of the hTDG. In addition to uracil and epsilonC-DNA glycosylase activity, the dsUDG protein repairs thymine in a G/T mismatch. The fact that epsilonC is recognized and efficiently excised by the E. coli dsUDG and hTDG proteins in vitro suggests that these enzymes may be responsible for the repair of this mutagenic lesion in vivo and be important contributors to genetic stability.

Figure 1

Cleavage of oligonucleotides containing ɛC residues by E. coli cells extract. 5′-end or 3′-end ³²P-labeled oligonucleotides containing ɛC residues were incubated with E. coli AB1157 cell extract. The products of the reaction were separated on a 20% denaturing PAGE. Lane 1, 5′-end ³²P-labeled ɛC/G duplex oligonucleotide. Lane 2, as lane 1, but incubated with 5 μg of E. coli crude extracts for 10 min. Lane 3, 3′-end ³²P-labeled ɛC/G duplex oligonucleotide. Lane 4, same as lane 3, but incubated with 5 μg of crude extracts for 10 min. The products of the reaction were analyzed by electrophoresis on a denaturing 20% polyacrylamide gel and visualized by using the PhosphorImager (model Storm 840). For details see Materials and Methods.

Figure 2

Distribution of ɛC and uracil-DNA glycosylase activities after FPLC chromatography on Mono S FPLC HR 5/5 column. The proteins containing the ɛCDG activity (fraction IV) were loaded on a Mono S FPLC HR 5/5 column and eluted by a linear NaCl gradient. (A) 250 fmol of the ɛC-34/G (■) duplex oligonucleotides were incubated with 1 μl from each column fraction for 10 min in a 50-μl reaction mixture. (B) 250 fmol of the U-34/G (▴) duplex oligonucleotides were incubated with 1 μl from each column fraction for 30 min in a 50-μl reaction mixture. The products of the reaction was analyzed as described in Fig. 1 and quantified by using the PhosphorImager (model Storm 840). For details see Materials and Methods.

Figure 3

Activity of the E. coli dsUDG protein using as substrate ɛC-34/G (■), U-34/G (▴,) or T-19/G (•, Inset) as substrate. 5′-³²P-labeled duplex oligonucleotide containing ɛC-34/G (■), U-34/G (▴), or T-19/G (•) mismatches (1 pmol in 100-μl reaction mixture) was incubated with increasing amounts of pure dsUDG protein for different periods of time in the presence of the Fpg protein. The products of the reaction were analyzed and quantified as described in Fig. 2. Each point represents the initial velocity of the enzymatic reaction. For details see Materials and Methods. Note the difference of scales in the Inset.

Figure 4

Action of various E. coli and human DNA repair proteins on the 34-mer duplex ɛC/G oligonucleotide. The 5′-³²P-labeled ɛC-34/G was incubated with an excess of the various pure repair protein at 37°C for 30 min (unless otherwise stated). Except for the control ɛC-34/G oligonucleotide and this oligonucleotide treated with Nth, Nfo, or Xth protein, the reactions were made in the presence of Fpg protein (50 ng) to reveal any abasic site generated by DNA glycosylases devoid of β-lyase activity. Lane 1, control ɛC-34/G oligonucleotide. Lane 2, as lane 1, but treated by E. coli dsUDG protein (5 ng). Lane 3, hTDG (150 ng, 30°C). Lane 4, AlkA (400 ng). Lane 5, ANPG40 (1.3 μg). Lane 6, Fpg protein (1 μg). Lane, 7, Nth protein (100 ng). Lane 8, Nfo (1.2 μg). Lane 9, Xth (4 nM, 10 min, 23°C). Lane 10, UNG (85 ng). Lane 11, APDG60 protein (1 μg). Lane 12, Tag I (350 ng). Lane 13, control as 1. The products of the reaction were analyzed as described in Fig. 1. For details see Materials and Methods.

Figure 5

Mechanism of action of the dsUDG and hTDG proteins on ɛC/G oligonucleotide. The ɛC-34/G duplex oligonucleotide was incubated with dsUDG protein or hTDG protein, and subsequently treated or not with proteins nicking at AP sites to reveal abasic sites generated by dsUDG or hTDG proteins. The 5′-³²P-labeled 34-mer ɛC-34/G duplex oligonucleotide is: lane 1, incubated at 37°C for 30 min; lanes 2, 6, and 10, incubated with 100 ng of Fpg protein at 37°C for 10 min; lanes 3, 7, and 11, incubated with 100 ng of Nth protein at 37°C for 10 min; lanes 4, 8, and 12, incubated with 100 ng of Nfo protein at 37°C for 10 min; lanes 5–8, incubated with 2 ng of dsUDG protein at 37°C for 10 min; lanes 9–12, incubated with 50 ng of hTDG protein at 30°C for 30 min. The products of the reaction were analyzed as described in Fig. 1. Arrow A indicates the 19-mer oligonucleotide containing an α,β-unsaturated aldehyde at the 3′-end. Arrow B indicates the 19-mer oligonucleotide containing a phosphate at the 3′-end. Arrow C indicates the 19-mer oligonucleotide containing 3′-OH termini. Arrow D indicates the 34-mer ɛC-34 oligonucleotide. For details see Materials and Methods.