Participation of mouse DNA polymerase iota in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer

Abstract

DNA polymerase iota (pol iota) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol iota performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol eta, pol iota, or both. A deficiency in mouse DNA polymerase eta (pol eta) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol iota, indicating that pol iota participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol eta- and pol iota-deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol iota status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol eta, pol iota, or both. Although pol iota deficiency alone had no effect, UV-induced skin tumors in pol eta-deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol iota. Collectively, these data reveal functions for pol iota in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer.

Fig. 1.

Survival of primary mouse fibroblasts after UV irradiation. The influence of mouse genotype on survival of primary dermal fibroblasts after irradiation with UV_{254 nm} was measured by colony-forming ability, as described in Methods. The fluence required to reduce the survival of Polη^+/+/ι^+/+ cells to 37% of the unirradiated control (D₃₇) was 8 J/m². This was reduced to ≈2.8 J/m² in the cells derived from Polη^−/−/ι^+/+. In both Polη^+/+ and Polη^−/− backgrounds, disruption of Polι slightly reduced UV survival. The significance of the reduced survival of pol ι-deficient cells was determined by polynomial regression analysis (53), with the best fit provided by cubic polynomials. This analysis revealed that the percent survival for Polη^+/+/ι^−/− cells was significantly lower than for Polη^+/+/ι^+/+ cells (P = 0.018), and that the percent survival for Polη^−/−/ι^−/− cells was significantly lower than for Polη^−/−/ι^+/+ cells (P = 0.001).

Fig. 2.

Frequency of TG-resistant (TG^r) clones as a function of survival after UV irradiation. Cells were plated on three 150-mm-diameter dishes at a density of 10⁴ cells per dish to determine mutant frequency or at cloning density to determine survival. After attachment, plates were irradiated with UV fluences estimated from the data in Fig. 1 to yield 20–40% survival. The actual survival in the mutagenesis experiments was determined as in Fig. 1 and plotted on the x axis. The corresponding mutant frequency at each survival is plotted on the y axis. Each point represents the mean of three independent dishes at the indicated survival, ±1 SD. Mutant frequency is defined as the number of TG^r clones per million clonable cells. Each data point represents independent experiments in which 2–4 × 10⁶ surviving cells were selected after UV irradiation and an 8- to 9-day expression period. The data have been corrected for cloning efficiency on the day of selection, and the spontaneous background mutant frequency (1 × 10⁻⁵) has been subtracted. The arrows indicate the reduction in mutant frequency when Polι is disrupted in the pol η-deficient background (larger arrow) and in the pol η-proficient background (smaller arrow).

Fig. 3.

Strand specificity of UV-induced mutagenesis. The strand containing the dipyrimidine sequence was determined in the case of each targeted base substitution. The proportion of mutations that arose from putative photoproducts in either strand was multiplied by the mutant frequency induced at D₃₇ for each of the four genotypes as presented in Fig. 2.

Fig. 4.

UV light-induced skin cancer in mice. Mice were shaved once per week and irradiated three times per week with 3.75 kJ/m² for 20 weeks or until the first skin tumor arose. Mice were inspected weekly for the development of skin tumors. Results with 12 Polη^−/−/ι^−/− mice (open circles) are compared with results reported (22) for 14 wild-type mice (open squares) and 12 homozygous Polη knockout mice (open diamonds), eight of which were Polι^+/−, and four of which were Polι^+/−. We also irradiated 13 Polη^+/−/ι^−/− mice and nine Polη^+/+/ι^−/− mice, none of which developed skin tumors after 20 weeks of irradiation (not plotted). In fact, the latter mice did not develop skin tumors after >41 weeks.