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Abstract

High DNA replication fidelity is achieved by the interplay of DNA polymerase nucleotide selectivity and proofreading activity and the DNA mismatch repair (MMR) system. Moreover, the overall concentration and the balance between the different dNTPs influence DNA polymerase fidelity. Consequently, deregulations in any of these four processes are frequently associated to increased mutagenesis and cancer susceptibility. This work addresses first, whether additional previously unrecognized genes support DNA replication fidelity and second, how altered dNTP pools impact on DNA replication fidelity in Saccharomyces cerevisiae. To identify previously unrecognized genes that prevent the accumulation of mutations, the budding yeast non-essential gene deletion collection was screened for increased mutagenesis in the presence of either the WT or low-fidelity DNA polymerase active-site mutants used as “sensitized mutator backgrounds”. This screen identified that loss of the folylpolyglutamate synthetase Met7 caused an increased mutator phenotype as well as increased gross chromosomal rearrangements (GCRs). GCRs were driven in large by dUTP accumulation and processing of uracil misincorporated into genomic DNA. Further characterization revealed that the accumulation of uracil alone is not sufficient to cause GCRs in budding yeast suggesting that GCRs in the absence of Met7 are the combined result of uracil accumulation and a DNA double-strand break repair defect. The genome-wide screen also revealed a group of genes that become critically important if DNA replication fidelity is compromised. Loss of either the CTP synthetase Ura7 or glutamine deficiency due to the absence of the transcription factor Gln3, resulted in reduced de novo CTP production. This alteration in the dNTP precursor pool caused a severe dNTP imbalance with a high mutagenic potential for which neither the ribonucleotide reductase (RNR) nor any mechanism downstream RNR could compensate. Thus, this study highlights the importance of the dNTP precursor metabolism on dNTP homeostasis and DNA replication fidelity and suggests that low CTP/dCTP pools are the Achilles’ heel of dNTP pool regulation. To investigate the effect of different dNTP pool alterations on DNA replication fidelity a RNR1 random mutagenesis screen was performed. The screen revealed key residues in RNR1, the large subunit of RNR, with crucial functions for dNTP homeostasis. The identified rnr1 alleles caused highly mutagenic dNTP alterations with different dependencies on DNA proofreading and MMR. dNTP imbalances characterized by one limiting dNTP facilitated not only base pair substitutions, but also frameshift mutations. In the subset of the identified dNTP alterations, the ones with low dATP and strongly elevated dGTP pools were most detrimental for DNA replication fidelity causing strong mutator phenotypes even in the presence of WT DNA polymerases and MMR. Taken together, this study highlights the pivotal role of the cellular metabolism and dNTP pool homeostasis on DNA replication fidelity. The identified genes and conditions may play a role as mini-drivers during tumor evolution and potentially represent future drug targets or prognostic markers.