A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance

Abstract

The balance and the overall concentration of intracellular deoxyribonucleoside triphosphates (dNTPs) are important determinants of faithful DNA replication. Despite the established fact that changes in dNTP pools negatively influence DNA replication fidelity, it is not clear why certain dNTP pool alterations are more mutagenic than others. As intracellular dNTP pools are mainly controlled by ribonucleotide reductase (RNR), and given the limited number of eukaryotic RNR mutations characterized so far, we screened for RNR1 mutations causing mutator phenotypes in Saccharomyces cerevisiae. We identified 24 rnr1 mutant alleles resulting in diverse mutator phenotypes linked in most cases to imbalanced dNTPs. Among the identified rnr1 alleles the strongest mutators presented a dNTP imbalance in which three out of the four dNTPs were elevated (dCTP, dTTP and dGTP), particularly if dGTP levels were highly increased. These rnr1 alleles caused growth defects/lethality in DNA replication fidelity-compromised backgrounds, and caused strong mutator phenotypes even in the presence of functional DNA polymerases and mismatch repair. In summary, this study pinpoints key residues that contribute to allosteric regulation of RNR’s overall activity or substrate specificity. We propose a model that distinguishes between different dNTP pool alterations and provides a mechanistic explanation why certain dNTP imbalances are particularly detrimental.

SAMHD1‐deficient fibroblasts from Aicardi‐Goutières Syndrome patients can escape senescence and accumulate mutations

“…14 Here, we found a similar situation even in non-cancer cells. 14 The high mutation frequency in SAMHD1deficient fibroblasts is likely related to the nature of their pool imbalance, that is, enlarged pools with very high dGTP, similar to the most mutagenic imbalance described by Schmidt et al 58 On this basis, we cannot exclude that an accumulation of somatic mutations in AGS cells may have contributed to the ability of P1 and P2 fibroblasts to proliferate in culture. Downregulation of MLH1 in AGS cells resulted in almost twofold increase in the mutation frequency.…”

“…Their large and imbalanced dNTP pools are expected to reduce the fidelity of DNA polymerases by interfering with their selectivity and proofreading activity 23,58 and by favoring mismatch extension. Their large and imbalanced dNTP pools are expected to reduce the fidelity of DNA polymerases by interfering with their selectivity and proofreading activity 23,58 and by favoring mismatch extension.…”

A targeted next generation sequencing approach to develop robust, genotype-specific mutation profiles and uncover novel variants inSaccharomyces cerevisiae

“…9). Notably, rnr1 alleles that dramatically increased dGTP levels were also severely mutagenic (rnr1K243E and rnr1I262V, N291D), increasing SNVs and frame shift mutations in repetitive contexts (SCHMIDT et al 2019). This highlights the importance of proper absolute and relative abundance of dGTP.…”

“…Quantification of dNTP levels in HeLa cell lines demonstrated elevated levels of all four dNTPs with an approximate 2-fold increase in dATP and dCTP and a 4-fold increase in dTTP and dGTP (MATHEWS 2015). Different skewed elevations result in distinct mutation spectra (SCHMIDT et al 2019)…”

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

“…MLH2 random mutagenesis screen. The plasmid library of randomly mutagenized MLH2 was generated by mutagenic PCR amplification and in vivo gap repair in the lig4Δ strain HHY6620 that is deficient in non-homologous end joining, similar as previously described 22 . Transformants were plated on SD plates lacking uracil (Ura − ) and then replica-plated onto frameshift mutator reporter plates (SD Ura − Lys − and SD Ura − Thr − to test for increased lys2-10A and hom3-10 frameshift reversion mutations, respectively); and onto SD Ura − Arg − + 60 mg/L canavanine to identify CAN1 inactivation mutations 8,32 .…”