Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio

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.

Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples

“…The rationale behind selecting an embryonal extract for these studies was that it represents an average sampling of the cell types in the body and therefore gives better coverage of additional peaks to consider when optimizing an HPLC protocol for general use. The dUTP levels were undetectable in the embryonal extract, which is in line with our previous observations in S. cerevisiae that dUTP is only detectable if enzymes involved in its metabolism are mutated ( 29 ). We also created a second HPLC protocol termed the Fast Protocol.…”

Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function

“…Lysates were analyzed on 8% or 10% SDS-PAGE followed by immunoblotting. The following antibodies were used: anti-HA (1:5,000, 3F10, Roche), anti-c-Myc (1:1,000, 4A6, Millipore), anti-Sic1 (1:10,000) 53 , anti-Pgk1 (1:20,000, 22C5D8, Invitrogen), anti-hMLH1 (1:1,000, BD-551091), anti-hPMS1 (1:1,000, sc-615, Santa Cruz), and anti-actin (1:5,000, A2228, Sigma). Western blots were developed using Immobilon Western Chemiluminscent HRP substrate (Millipore) and imaged using Super RX-N Fuji medical X-ray films (Fujifilm) or using Fusion Solo S (Vilber).…”

Vitamins as Possible Cancer Biomarkers: Significance and Limitations

“…The increased DNA damage induced by folate depletion is applied in cancer therapy. Antifolate drugs are administered purposely to induce DNA insult in the fast-replicating cancer cells [99], highlighting the importance of this micronutrient for the chromosomal stability of the cells. On the other hand, the depletion of methylcytosine (C me ) triggered by the depletion of vitamin B 9 induces a global DNA demethylation that can foster oncogenesis [100].…”