Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease

Abstract

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N:-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF-, AAF- and AP-DNA adducts, determined by gel mobility shift assay, are 33 +/- 9, 8 +/- 2 and 23 +/- 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF-DNA was cleaved approximately 2-fold more efficiently than AF- or AP-DNA (AAF > AF approximately AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF-DNA (but not for AP-DNA), making it equivalent to that for AAF-DNA. These results are consistent with a model in which DNA damage recognition by the E. coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF- and AAF-DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.

Figure 1

DNA adducts and substrates used in the present study. (A) Chemical structures of 2-aminofluorene (AF)-, N-acetyl-2-aminofluorene (AAF)- and 1-nitropyrene-derived 1-aminopyrene (AP)-C8-dG DNA adducts. (B) Substrates constructed for this study.

Figure 2

Binding of UvrA to AF–, AAF– and AP–DNA substrates. UvrA at the concentrations specified was incubated at 37°C for 10 min with 1 nM AF–, AAF– or AP–DNA substrates (B0) in UvrABC buffer and then analyzed on a 4% polyacrylamide native gel in a gel mobility shift assay.

Figure 3

Incision of AF–, AAF– and AP–DNA adducts by UvrABC nuclease. (A) The 5′-terminally labeled DNA substrates (B0, 3 nM) containing an AF, AAF or AP adduct were incubated with UvrABC (10 nM UvrA, 250 nM UvrB and 100 nM UvrC) in UvrABC buffer at 37°C for the period indicated. The incision products were then analyzed on a 12% polyacrylamide sequencing gel. (B) Incision kinetics of the DNA substrates incised by UvrABC. Data represent the means ± SD of at least three independent experiments.

Figure 3

Incision of AF–, AAF– and AP–DNA adducts by UvrABC nuclease. (A) The 5′-terminally labeled DNA substrates (B0, 3 nM) containing an AF, AAF or AP adduct were incubated with UvrABC (10 nM UvrA, 250 nM UvrB and 100 nM UvrC) in UvrABC buffer at 37°C for the period indicated. The incision products were then analyzed on a 12% polyacrylamide sequencing gel. (B) Incision kinetics of the DNA substrates incised by UvrABC. Data represent the means ± SD of at least three independent experiments.

Figure 4

Incision of AF–, AAF– and AP–DNA bubble substrates by UvrABC. (A) 5′-Terminally labeled DNA bubble substrates (B6, 3 nM) containing an AF, AAF or AP adduct were incubated with UvrABC (10 nM UvrA, 250 nM UvrB and 100 nM UvrC) in UvrABC buffer at 37°C for the period indicated. The incision products were then analyzed on a 12% polyacrylamide sequencing gel. (B) Incision kinetics of the DNA substrates incised by UvrABC. Data represent the means ± SD of at least three independent experiments.

Figure 4

Incision of AF–, AAF– and AP–DNA bubble substrates by UvrABC. (A) 5′-Terminally labeled DNA bubble substrates (B6, 3 nM) containing an AF, AAF or AP adduct were incubated with UvrABC (10 nM UvrA, 250 nM UvrB and 100 nM UvrC) in UvrABC buffer at 37°C for the period indicated. The incision products were then analyzed on a 12% polyacrylamide sequencing gel. (B) Incision kinetics of the DNA substrates incised by UvrABC. Data represent the means ± SD of at least three independent experiments.