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.DNA replication fidelity | mismatch repair | CTP biosynthesis | DNA polymerases | dNTP pool imbalance T he fidelity of DNA replication is strongly influenced by three processes (1-3): (i) nucleotide selectivity, wherein replicative DNA polymerases select the correct dNTP to be incorporated; (ii) DNA polymerase proofreading activity, which excises wrongly incorporated nucleotides by using the DNA polymerase 3′-to-5′ exonuclease activity; and (iii) mismatch repair (MMR) (4, 5), a DNA replication-coupled repair mechanism (6, 7), which corrects errors that escaped proofreading. Furthermore, the balance and overall concentration of dNTPs not only affect nucleotide selection but also influence DNA polymerase proofreading activity (8). A central player in the biosynthesis of dNTPs is the ribonucleotide reductase (RNR) holoenzyme, which catalyzes the reduction of NDPs to dNDPs (9, 10). In Saccharomyces cerevisiae, RNR is composed of two identical Rnr1 large subunits that associate with two smaller subunits represented by Rnr2 and Rnr4 (11,12). In addition, a second large subunit has been identified (Rnr3), which is induced upon DNA damage and when overexpressed can rescue the rnr1 lethal phenotype (13).The interplay between nucleotide selectivity, DNA polymerase proofreading activity, and MM...

DNA polymerase δ proofreads errors made by DNA polymerase ε

“…Haploid yeast deficient in Polδ proofreading require functional MMR for survival (26). Recent work has demonstrated that polymerase fidelity and MMR can compensate for defects in cellular metabolism that lead to dNTP pool imbalances and help maintain a normal low mutation rate despite the abnormal dNTP levels (60,61). Extrinsic proofreading of Pole errors by Polδ shown here as well as proofreading of Polα errors by Polδ shown previously (9) is yet another mechanism of redundancy to prevent accumulation of DNA replication errors.…”

Fast and sensitive HPLC–MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells

“…dNTP pools are tightly controlled and an imbalance of dNTPs is observed in a variety of disease conditions such as cancer and mitochondrial diseases[1,2]. Both imbalanced dNTP pools as well as irregularly large amounts of all dNTPs have been shown to be associated with increased errors in nucleotide selectivity combined with inefficient proofreading during DNA replication and eventual oncogenesis, likely attributed to this buildup of mutations[3,4]. During the cell cycle, dNTP pools are augmented for DNA synthesis and are generally elevated during proliferation [1,5].…”

DNA replication and mismatch repair safeguard against metabolic imbalances

“…Mutations in genes encoding MMR proteins and DNA polymerase proofreading activities are linked to elevated mutation rates and diseases such as cancer, and these rates are synergistically increased in double mutants (2,3). In PNAS, Schmidt et al (4) describe work in which they screened for defects in genome stability in genetic backgrounds where processes that limit mutagenesis were compromised. Their work provides new insights for how cellular metabolism and nucleotide pool homeostasis interact to avoid mutation and maintain genome stability.…”

“…Previous screens of this type, performed in functional DNA polymerase backgrounds, identified genome stability factors that are likely to be important targets in cancer (5-7). Schmidt et al (4) used this approach to identify new targets whose effects on mutation rate are normally buffered by the fidelity of DNA polymerases. Their approach identified knockout mutations in the EXO1, RRM3, GLN3, URA7, and SHM2 genes.…”

“…Furthermore, for four of the five knockouts (exo1 knockout was the exception), the mutator phenotype seen in the DNA polymerase active-site mutant backgrounds was detected only in the canavanine resistance assay. Because elevated mutation rates were not observed in the Lys + reversion assay, Schmidt et al (4) surmised that the mutator phenotypes seen in polymerase mutants lacking RRM3, GLN3, URA7, and SHM2 were likely the result of base substitutions; they confirmed this hypothesis by analyzing mutation rates in MMR-deficient backgrounds that have strong defects in the repair of base-base mismatches (msh6 knockout). This work also encouraged them to understand the roles of the GLN3, URA7, and SHM2 gene products better.…”