Abstract
The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation. Here, we found that Met7-deficient strains show elevated mutation rates, but also increased levels of endogenous DNA damage resulting in gross chromosomal rearrangements (GCRs). Quantification of deoxyribonucleotide (dNTP) pools in cell extracts from met7Δ mutant revealed reductions in dTTP and dGTP that cause a constitutively active DNA damage checkpoint. In addition, we found that the absence of Met7 leads to dUTP accumulation, at levels that allowed its detection in yeast extracts for the first time. Consequently, a high dUTP/dTTP ratio promotes uracil incorporation into DNA, followed by futile repair cycles that compromise both mitochondrial and nuclear DNA integrity. In summary, this work highlights the importance of folate polyglutamylation in the maintenance of nucleotide homeostasis and genome stability.
Highlights
The one-carbon (1C) cycle is a central metabolic pathway that comprises several modification reactions of folates, which are used as 1C donors in a variety of biosynthetic processes
Among the genes that prevented frameshift mutations, we identified known components of the mismatch repair (MMR) system (MSH2, MSH6, MSH3, MLH1, MLH3, PMS1 and EXO1) that participate at different steps during the correction of insertions/deletions or base substitutions [49,50,51]
After analyzing cell lysates from logarithmically growing WT and met7Δ strains by western blotting, we found that loss of Met7 resulted in phosphorylation of the DNA damage checkpoint kinase Rad53, and up-regulation of the DNA damage-inducible ribonucleotide reductase (RNR) subunit Rnr3 that contributes to dNTP biosynthesis (Figure 1A)
Summary
The one-carbon (1C) cycle is a central metabolic pathway that comprises several modification reactions of folates, which are used as 1C donors in a variety of biosynthetic processes. Folate cofactors are required for dTMP and purine biosynthesis (Supplementary Figure S1), glycine/serine homeostasis, homocysteine remethylation to methionine and the production of formyl-methionyl-tRNA that is necessary for the initiation of protein biosynthesis in bacteria, chloroplasts and mitochondria [1,2]. Due to the pivotal role of the 1C metabolism for cell proliferation and growth, drugs that target the 1C cycle (antifolates) have proved beneficial for treatment of cancer, autoimmune chronic diseases, as well as bacterial and parasite infections [3,4,5,6,7]. Antifolates currently in use for cancer treatment inhibit dihydrofolate reductase (DHFR), that converts 7,8-dihydrofolate (DHF) into tetrahydrofolate (THF), the glycinamide ribonucleotide formyltransferase (GARFT) that uses 10-formyl-THF during the synthesis of purines, and thymidylate synthase (TS) that catalyzes the conversion of 2-deoxyuridine monophosphate (dUMP) into dTMP [8]. Since eukaryotic DNA replicative polymerases cannot distinguish between dTTP and dUTP [16], an in-
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