Human mitochondrial DNA polymerase γ exhibits potential for bypass and mutagenesis at UV-induced cyclobutane thymine dimers

Abstract

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3'-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3'-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.

FIGURE 1.

Replication bypass of thymine dimers catalyzed by human DNA pol γ. The substrates used for the assay (standing start, lanes 1–8 and 17–24, or running start, lanes 9–16 and 25–32) are represented schematically on the top of each autoradiogram, with the asterisk indicating the radiolabeled strand. Primer extension reactions were carried out with 50 nm substrate and 10 nm pol γ holoenzyme at 37 °C for 10 min with varying concentrations of dNTPs as described under “Experimental Procedures.” Lanes 1–16 and 17–32 contain exonuclease-deficient and exonuclease-proficient pol γ holoenzyme, respectively. Lanes 1–4, 9–12, 17–20, and 25–28, non-damaged substrate (ND). Lanes 5–8, 13–16, 21–24, and 29–32, thymine dimer substrate (T-T). Lanes 1, 5, 9, 13, 17, 21, 25, and 29, no dNTP. Lanes 2, 3, and 4 contain 0.01, 0.04, and 0.16 μm dNTPs, respectively. Lanes 10–12 contain the same concentrations as lanes 2–4. Lanes 18, 19, and 20 have 0.1, 0.4, and 1.6 μm dNTPs, respectively. Lanes 26–28 have the same concentrations as lanes 18–20. Lanes 6, 7, and 8 contain 10, 40, and 160 μm dNTPs, respectively. Lanes 14–16, 22–24, and 30–32 contain the same concentrations as lanes 6–8.

FIGURE 2.

Single-nucleotide incorporation catalyzed by exonuclease-deficient DNA pol γ opposite the dimer-containing templates. Schematic representation of the substrate used with the location of the thymine dimer is shown on the top of the autoradiogram with the asterisk denoting the radiolabeled strand. Incorporation opposite the T-T dimer was performed as described under “Experimental Procedures.” Lanes 1–5, non-damaged substrate (ND); lanes 6–10, thymine dimer substrate (T-T); lanes 1 and 6, no dNTP; lanes 2 and 7 contain 0.04 and 40 μm dATP, respectively; lanes 3 and 8 contain 10 and 100 μm dGTP, respectively; lanes 4 and 9 contain 200 μm dCTP; lanes 5 and 10 contain 50 and 100 μm dTTP, respectively. The concentration of each dNTP used in this representative gel was determined based on the K_m values from Table 1.

FIGURE 3.

Single-nucleotide extension analysis of exonuclease-deficient DNA pol γ past the thymine dimers in template. Schematic representation of the substrate used with the location of the thymine dimer is shown on the top of the autoradiogram with the asterisk denoting the radiolabeled strand. Extension reactions past the T-T dimer was carried out as described under “Experimental Procedures.” Lanes 1, 2, 5, 6, 9, and 10, ND, non-damaged substrate; lanes 3, 4, 7, 8, 11, and 12, T-T, thymine dimer substrate; lanes 1, 3, 5, 7, 9, and 11, no dNTP; lanes 2 and 4 contain 0.04 and 0.8 μm dCTP, respectively; lanes 6, 8, 10, and 12 contain 0.04, 40, 0.32, and 40 μm dATP, respectively. The concentration of each dNTP used in this representative gel was determined based on the K_m values from Table 3.

FIGURE 4.

Primer extension by exonuclease-deficient human DNA pol γ from various 3′-end terminated primers annealed to thymine dimer-containing templates. The substrate used for the assay is represented schematically on top of each autoradiogram with the asterisk indicating the radiolabeled strand. Primer extension assays were carried out as described under “Experimental Procedures.” −, no dNTP. Lanes 2, 3, and 4 contain 0.01, 0.04, and 0.16 μm dNTPs, respectively. Lanes 10–12, 18–20, and 26–28 contain the same concentrations as lanes 2–4. Lanes 6, 7, and 8 contain 10, 40, and 160 μm dNTPs, respectively. Lanes 22–24 and 30–32 contain the same concentrations as lanes 6–8. Lanes 14, 15, and 16 have 0.5, 2, and 8 μm dNTPs, respectively.