Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis

Abstract

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in Saccharomyces cerevisiae using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (GLN3, URA7, and SHM2) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.

Keywords: CTP biosynthesis; DNA polymerases; DNA replication fidelity; dNTP pool imbalance; mismatch repair.

Fig. 1.

Genome-wide screen identifies genes that affect DNA replication fidelity in S. cerevisiae. (A) Strategy used to cross the nonessential gene deletion collection with active-site DNA polymerase mutants. (B) To screen for mutator phenotypes in 96-well format, strains were spotted on YPD, grown, and replica plated on mutator reporter plates. Increased number of colonies is indicative of a potential mutator phenotype. On the right side (zoom-in), msh6Δ results in increased frameshifts (lysine−) and CAN1 mutations (+canavanine), whereas ubc13Δ increases CAN1 mutations, exclusively.

Fig. S1.

Representative pictures of reporter plates (zoom-in) illustrating mutator phenotypes in some S. cerevisiae double-mutant strains. Inactivation of Exo1 in lagging-strand DNA polymerase mutant backgrounds (pol1-L868M or pol3-L612M) results in frequent CAN1 inactivating mutations and frameshifts in lys2-10A allele, indicated by the higher abundance of Can^R and lysine⁺ colonies, respectively. Similarly, inactivation of Gln3 or Shm2 in lagging-strand DNA polymerase mutant backgrounds, results in increased CAN1 inactivation, but not frameshifts.

Fig. 2.

Inactivation of URA7 in Pol3 proofreading-defective background (pol3-01) results in severe growth defects and synergistic increases in mutation rates. (A) Plasmid shuffling strains pol3Δ, pol3Δ ura7Δ, and pol3Δ msh2Δ [all haploids (n) complemented with a WT POL3-URA3 plasmid] were transformed with either WT POL3 or pol3-01 LEU2-plasmids. Transformants were grown on Ura⁻ Leu⁻ SD plates or 5-FOA–containing plates to select against WT POL3-URA3 plasmid. Double-mutant combination msh2Δ + pol3-01 serves as positive control for a synthetic lethal interaction. (B) Haploid (n) or diploid homozygous (2n) pol3Δ ura7Δ mutants expressing either WT POL3 or mutant pol3-01, were grown as in A. (C) Proliferation curves. Diploid homozygous pol3Δ or pol3Δ ura7Δ strains were transformed with either WT POL3 or pol3-01 plasmids. Three independent isogenic strains for each genotype were grown overnight in YPD and diluted next day to OD₆₀₀ = 0.1 in fresh YPD. Proliferation was followed by OD₆₀₀ measurements, and the values were plotted as mean ± SD on log₂ scale. (D) Quantification of CAN1 inactivation rates in diploid strains hemizygous for CAN1 locus (SI Materials and Methods for additional details) and homozygous for pol3Δ or pol3Δ ura7Δ mutations complemented with POL3 or pol3-01 plasmids. Error bars represent the 95% confidence intervals (CIs) and numbers on top indicate the fold increase in the mutation rate over the WT diploid strain (2.4 × 10⁻⁷ Can^R mutants per cell division).

Fig. 3.

Inactivation of Ura7 or Gln3 results in DDR checkpoint activation. (A) Simplified diagram depicting DDR response in S. cerevisiae. (B) Whole-cell lysates of logarithmically growing cells were analyzed by Western blotting with Rad53 and RNR1-4 antibodies. WT cells treated with 200 mM hydroxyurea (HU) were used as control for activation of DDR. (C) DNA content profiles of the indicated strains. (D) Mutation rates in mutant strains in the presence or absence of DUN1. See also Table S3. (E) Mutation rates in the indicated strains grown in YPD media supplemented or not with 5 mM glutamine (Gln). Error bars represent the 95% CI, and numbers on top indicate the fold increase in the mutation rate over WT.

Fig. 4.

Inactivation of Gln3 or Ura7 results in NTP and dNTP imbalance causing increased G:C-to-A:T transitions. (A) NTP and (B) dNTP pool measurements in the indicated strains (Tables S4 and S5). Error bars represent SD. (C) Independent Can^R clones (n ≥ 91 per genotype) were sequenced for CAN1 mutations. Graphs indicate the type of identified mutations, in percentages (see also Tables S6 and S7). (D) The G-to-A mutational hotspot at nucleotide 788 was frequently found in msh6Δ gln3Δ, msh6Δ shm2Δ, and msh6Δ ura7Δ strains. Predicted mutation is noted in red. Nucleotides marked in green are facilitating mispair rapid extension prior proofreading due to their higher abundance in gln3Δ or ura7Δ mutants, compared with the WT strain. (E) The G-to-A mutational hotspot at position 497 was frequently found in msh6Δ and msh6Δ shm2Δ, but not in msh6Δ gln3Δ and msh6Δ ura7Δ. Here, the predicted G-dT mispair is immediately followed by incorporation of dCTP (in black), which is less abundant in gln3Δ and ura7Δ strains and, therefore, unlikely to support mispair rapid extension.

Fig. 5.

(A) Pathways of de novo dNTP biosynthesis in S. cerevisiae (adapted from ref. 9). Essential genes are shown in bold (RNR1, 2, and 4 are nonessential in certain yeast genetic backgrounds). Metabolism-related genes (GLN3, URA7, and SHM2) identified in this screen were encircled in red. (B) Gln3 and Ura7 promote DNA replication fidelity by preventing dNTP pool imbalances. Inactivation of Gln3 or Ura7 results in low CTP/dCTP levels, triggering DNA damage checkpoint activation. Up-regulation of RNR subunits, instead of compensating low dCTP pools, creates a severe dNTP pool imbalance that, in combination with altered DNA polymerase functions or partial MMR defects, causes severe mutator phenotypes.