dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Abstract

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Keywords: DNA replication and repair; cancer; lethal mutagenesis; polymerase fidelity.

Fig. 1.

Two S-phase checkpoint responses in S. cerevisiae. The S-phase checkpoint is triggered in the DNA damage signaling pathway by replication protein A (RPA)-coated DNA (red circles) resulting from ssDNA repair intermediates, resected ends of double-stranded breaks, or collapsed replication forks. In the replication stress signaling pathway, the S-phase checkpoint is triggered by stalled replication forks (α, δ, ε; replicative DNA polymerases). The two signaling pathways converge at Mec1/Ddc2, which phosphorylates Rad53 with the help of Rad9 (DNA damage) or Mrc1 (replication stress). Phospho-Rad53 then activates Dun1, which, in turn, inactivates three repressors (Sml1, Crt1, and Dif1) of RNR. RNR generates dNDPs from NDPs, which are then converted to dNTPs by nucleoside diphosphate kinases. Higher dNTP concentrations facilitate DNA repair and replication fork restart. Blue lines are signals that increase dNTP pools. Red lines are signals that repress dNTP pools. Adapted from ref. .

Fig. 2.

Dun1 promotes Pol ε error-prone replication. (A) Dun1 dependence of pol2 mutator alleles. (Top) Viability measurements. Dun1 and dun1Δ strains were transformed with LEU2 plasmids carrying POL2, pol2-4, or pol2-4,M644G. Ten-fold serial dilutions of the resulting transformants were then plated onto FOA media to select for loss of the complementing POL2-URA3 plasmid and were photographed after 3 d at 30 °C. (Bottom) Spontaneous mutation rates determined from multiple independent fluctuation analyses of each strain. Error bars represent 95% confidence intervals. “X” indicates synthetic lethality. (B) pol2-4 and pol2-M644G mutator phenotypes do not require the translesion polymerase Pol ζ (rev3Δ) or Pol η (rad30Δ). Mutation rates were performed as described above. (C) Error-induced extinction in pol2-4 msh2Δ cells requires Dun1. (Top) Viability measurements. DUN1 and dun1Δ cells proficient for MMR, deficient in all MMR (msh2Δ), or deficient in only base–base MMR (msh6Δ) were transformed with POL2 or pol2-4 plasmids and plated on FOA using 10-fold serial dilutions. (Bottom) Mutation rates were determined as above.

Fig. 3.

Cell cycle progression and RNR transcript expression in pol2-4 and pol2-M644G strains. (A) Cell cycle progression of pol2 mutator strains. Cells were synchronized with α-factor, released into rich medium, and monitored for DNA content by FACS. The x axis represents relative DNA content, and the y axis represents cell number at the indicated time points. Vertical lines indicate ploidy (1C and 2C). (B) Checkpoint induction in pol2 mutator strains. Transcript levels of RNR1 and RNR3 were measured by quantitative PCR assay, normalized to actin expression, and then normalized to the levels found in POL2 control cells. RNA isolated from POL2 cells treated with 200 mM hydroxyurea for 2 h was used as a positive control for checkpoint activation. Error bars show 95% confidence intervals for transcript expression based on three independent biological replicas. Asterisks denote expression levels that are significantly different (P ≤ 0.05) from POL2 control cells.

Fig. 4.

Effects of Mrc1 and Rad9 on pol2-4 and pol2-M644G phenotypes. (A) Viability measurements. The mrc1Δ or rad9Δ strain was transformed with LEU2 plasmids carrying POL2, pol2-4, or pol2-M644G. Transformants were plated onto FOA media using 10-fold serial dilutions to select for loss of the complementing POL2-URA3 plasmid and photographed after 3 d at 30 °C. (B) Spontaneous mutation rates. Can^r mutants per cell division were determined from multiple independent fluctuation analyses of each strain. The 95% confidence intervals for each mutation rate are shown as error bars. In the grid below the graphs, black boxes designate strain genotypes.

Fig. 5.

dNTP levels correlate with mutator phenotypes. (A) Viability measurements. Strains with the indicated genotypes were transformed with LEU2 plasmids carrying Pol ε alleles (POL2, pol2-4, or pol2-M644G), plated onto FOA media to select for loss of the complementing POL2-URA3 plasmid, and photographed after 3 d at 30 °C. (B) Spontaneous mutation rates. Can^r mutants per cell division were determined from multiple independent fluctuation analyses of each strain. The 95% confidence intervals for each mutation rate are shown as error bars. In the grid below the graphs, black boxes designate strain genotypes. (C) dNTP measurements. dNTPs were harvested from duplicate midlog-phase cultures of cells with the indicated genotypes, normalized to NTP levels in each sample, and then divided by total number of cells used for the preparation. Error bars reflect SE measurement based on the variance between the duplicate samples.