Quantitative analysis of the mutagenic potential of 1-aminopyrene-DNA adduct bypass catalyzed by Y-family DNA polymerases

Abstract

N-(Deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dG(AP) induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dG(AP) lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dG(AP), we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dG(AP). Opposite dG(AP) and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dG(AP). Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dG(AP) bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dG(AP) bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dG(AP) in humans.

Figure 1

Standing start assay for hRev1 at 37 °C. The sequence of damaged DNA substrate is shown above the gel image with the location of dG^AP (21^st position) is colored in red and in bold type. Each lane is a 10-min reaction. “0”, “A”, “C”, “G”, “T” and “4” denote the addition of no dNTPs, dATP (200 μM), dCTP (200 μM), dGTP (200 μM), dTTP (200 μM), and all four dNTPs (200 μM each), respectively. DNA substrates used were (A) 20-mer/26-mer and (B) 20-mer/26-mer-dG^AP.

Figure 2

Comparison of preferred actions by Dpo4, hPolη, hPolκ, and hPolι opposite dG^AP and an immediate downstream template position in the DNA substrate 17-merS/73-merAP or the corresponding template bases dG and dC in the control DNA substrate 17-merS/73-mer. The relative frequencies (%) of nucleotide misincorporations or deletions were calculated at template Position X (A) or 0 (B) and Position +1 (C and D) in the presence of dG^AP (A and C) or dG (B and D) for Dpo4 (black bar), hPolη (white bar), hPolκ (striped bar), and hPolι (light grey bar).

Figure 3

Histogram of relative error% produced by Dpo4 as a function of DNA template position. At each position along the DNA template, the relative base substitution% (white bar) and deletion% (black bar) are shown to reveal the total relative error% and the contribution of each type of mutations simultaneously. The dG^AP site is indicated as an “X” along the X-axis (A), and the corresponding template base dG is denoted as “0” along the X-axis (B).

Figure 4

Histogram of relative error% as a function of DNA template position for human Y-family DNA polymerases. At each position along the DNA template, the relative base insertion% (striped bar), substitution% (white bar) and deletion% (black bar) are shown to reveal the total relative error% and the contribution of each type of mutations simultaneously. The dG^AP site is indicated as an “X” along the X-axis, and the corresponding template base dG is denoted as “0” along the X-axis. The dG^AP bypass analysis is shown for hPolη (A), hPolκ (C), and hPolι, (E). DNA synthesis with the control 17-merS/73-mer was also analyzed for hPolη (B), hPolκ (D), and hPolι (F). Statistically significant differences (p < 0.05) of the relative total error rates between the SOSA products generated from the damaged and control DNA substrates at each template position are indicated by *.

Scheme 1