The mechanism of nucleotide excision repair-mediated UV-induced mutagenesis in nonproliferating cells

Abstract

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps.

Figure 1

Models for NER-associated mutagenesis. The NER machinery is recruited either by a UV-induced CPD or (6-4) photoproduct (yellow stars) or by an interstrand crosslink (|). NER excises an oligonucleotide either from the strand containing the lesion (models A, C, and D) or from the undamaged strand (model B). The resulting gap is filled by a DNA polymerase (Pol), which introduces a mutation (red “m”) opposite a lesion within the gap (models A, B, and D) or opposite an undamaged template (model C). Finally, the mutation is introduced into both DNA strands by a second round of NER (models A, B, and D) or by MMR (model C). The Pso2 protein is specifically required for the bypass of an interstrand crosslink. Newly synthesized DNA within NER or MMR-generated gaps is indicated by red dashed lines.

Figure 2

The ade2 adeX mutation system. (A) Relevant genes in the adenine biosynthetic pathway are indicated. A red pigment accumulates in the absence of either the ADE1 or the ADE2 gene product; production of the pigment is blocked by inactivation of any gene product that functions prior to Ade1/Ade2 in the pathway. PRPP, phosphoribosyl pyrophosphate; AIR, aminoimidazole ribotide; CAIR, carboxyaminoimidazole ribotide; IMP, inosine monophosphate. (B) Following DNA damage, pure white colonies are produced only if an adeX mutation is present in both strands of duplex DNA. Bypass of damage during DNA replication results in a red-white sectored colony.

Figure 3

Role of NER in the production of two-strand mutations in nondividing cells. Induction frequencies of white (open bars) and red-white sectored (red cross-hatched bars) ade2 adeX mutants in a G0 population of cells are shown.

Figure 4

Neither MMR nor ICLR is required for two-strand mutations in nondividing cells. Induction frequencies of pure white (open bars) and red-white sectored (red cross-hatched bars) ade2 adeX mutants in G0 populations of WT, mlh1Δ, and pso2Δ cells are shown.

Figure 5

Requirements of error-prone and error-free bypass pathway components for two-strand mutations in nondividing cells. Induction frequencies of pure white (open bars) and red-white sectored (red cross-hatched bars) ade2 adeX mutants in G0 populations of cells are shown. Asterisks above the bars indicate a lack of UV-induced mutagenesis (irradiated to unirradiated sample comparison, P > 0.1 in all cases).

Figure 6

Roles of checkpoint proteins in the production of two-strand mutations in nondividing cells. Induction frequencies of pure white (open bars) and red-white sectored (red cross-hatched bars) ade2 adeX mutants in G0 populations of cells are shown. Presence of sml1Δ is required for the viability of mec1Δ and rad53Δ strains and does not affect UV sensitivity. The asterisk above the rad17Δ bar indicates a lack of UV-induced mutagenesis (irradiated to unirradiated sample comparison, P > 0.8).

Figure 7

UV-induced killing and mutagenesis in a WT strain. (A) UV-induced killing. For each UV dose, survival was determined in three independent experiments; these values are plotted and overlap at each dose. The data are best fit (R² = 0.9986) with a quadratic killing curve (Qk, solid line) with the coefficient of lethality k₂ = 0.000389 (J/m²)⁻². Approximation of the data with a linear function (Lk) is represented as dotted lines. Approximation with a linear-quadratic function is not represented due to negative k₁ value. (B) Average values of UV-induced pure white ade2 adeX mutant frequencies (circles) and red-white sectored ade2 adeX mutant frequencies (squares) obtained in three independent experiments. (C) Yields of UV-induced pure white ade2 adeX mutants. For each UV dose, values obtained in three independent experiments (open circles) as well as average values (solid circles) are plotted. The data are best fit (R² = 0.9604) with a linear-quadratic mutagenesis curve (LQm, solid line) with the coefficients of mutability m₁ = 0.3503 (J/m²)⁻¹ and m₂ = 0.003015 (J/m²)⁻². Approximation of the data with a linear mutagenesis function (Lm) is represented as a dashed line [R² = 0.8907; m₁ = 0.5095 (J/m²)⁻¹]. Approximation with a quadratic function (Qm) is represented as a dotted line. (D) Frequencies of UV-induced pure white ade2 adeX mutants. For each UV dose, values obtained in three independent experiments (open circles) and average values (solid circles) are plotted. The data are best fit (R² = 0.9966) with a linear-quadratic mutagenesis curve (LQm, solid line) with the coefficients of mutability m₁ = 0.3503 (J/m²)⁻¹ and m₂ = 0.003015 (J/m²)⁻². Approximation of the data with a linear mutagenesis function (Lm) is represented as a dashed line [R² = 0.96; m₁ = 0.5095 (J/m²)⁻¹]. Approximation with a quadratic function (Qm) isrepresented as a dotted line. (E) Frequencies of UV-induced red-white sectored ade2 adeX mutants. For each UV dose, values obtained in three independent experiments (open squares) and average values (solid squares) are plotted. The data are well fit with a linear mutagenesis curve (R² = 0.9828).

Figure 8

UV-induced killing and mutagenesis in an NER-deficient (rad14Δ) strain. (A) UV-induced killing. Survival values obtained in three to five independent experiments (open symbols) and average values (solid symbols) are plotted. The data are best fit (R² = 0.9999) with a linear-quadratic killing curve (LQk, solid line) with coefficients of lethality k₁ = 0.3024 (J/m²)⁻¹ and k₂ = 0.1305 (J/m²)⁻². Approximation of the data with linear (Lk) or quadratic (Qk) functions is represented as dotted lines. (B) The average values of UV-induced pure white (circles) and red-white sectored ade2 adeX mutant frequencies (squares) obtained in three to five independent experiments are shown. (C) Yields of UV-induced pure white ade2 adeX mutants. For each UV dose, values obtained in three to four independent experiments (open circles) and average values (solid circles) are plotted. The data are best fit (R² = 0.9657) with a quadratic mutagenesis curve (Qm, solid line) with the coefficient of mutability m₂ = 1.455 (J/m²)⁻². An approximation of the data with a linear mutagenesis function (Lm) is represented as a dotted line. The approximation with a linear-quadratic function is not represented due to a negative m₁ value. (D) Frequencies of UV-induced pure white ade2 adeX mutants. For each UV dose, values obtained in three to four independent experiments (open circles) and average values (solid circles) are plotted. The data are best fit (R² = 0.9747) with a quadratic mutagenesis curve (Qm, solid line) with the coefficient of mutability m₂ = 1.455 (J/m²)⁻². Approximation of the data with a linear mutagenesis function (Lm) is represented as dotted line. An approximation with a linear-quadratic function is not shown due to a negative m₁ value. (E) Yields of UV-induced red-white sectored ade2 adeX mutants. For each UV dose, the values obtained in three to four independent experiments (open squares) and average values (solid squares) are plotted. The data are best fit (R² = 0.9503) with a linear mutagenesis curve (Lm, solid line) with the coefficient of mutability m₁ = 10.06 (J/m²)⁻¹. Approximation of the data with a quadratic mutagenesis function (Qm) is represented as dotted line. An approximation with a linear-quadratic function is not represented due to a negative m₁ value. (F) Frequencies of UV-induced red-white sectored ade2 adeX mutants. For each UV dose, values obtained in three to four independent experiments (open squares) and the average values (solid squares) are plotted. The data are best fit (R² = 0.9683) with a linear mutagenesis curve (Lm, solid line) with the coefficient of mutability m₁ = 10.06 (J/m²)⁻¹. Approximation of the data with a quadratic mutagenesis function (Qm) is represented as dotted line. An approximation with a linear-quadratic function is not shown due to a negative m₁ value.

Figure 9

Summary of one- and two-strand mutation frequencies in defined mutant backgrounds. (A) Genetic control of two-strand mutations. (B) Genetic control of one-strand mutations. Black symbols indicate WT frequencies. Frequencies similar to WT are plotted as blue symbols, those less than WT are in green, and those greater than WT are in red.