Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint

Abstract

A balanced supply of deoxyribonucleoside triphosphates (dNTPs) is one of the key prerequisites for faithful genome duplication. Both the overall concentration and the balance among the individual dNTPs (dATP, dTTP, dGTP, and dCTP) are tightly regulated, primarily by the enzyme ribonucleotide reductase (RNR). We asked whether dNTP pool imbalances interfere with cell cycle progression and are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. By introducing single amino acid substitutions in loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR, we obtained a collection of strains with various dNTP pool imbalances. Even mild dNTP pool imbalances were mutagenic, but the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity. The S-phase checkpoint was activated by the depletion of one or several dNTPs. In contrast, when none of the dNTPs was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression.

Figure 1.

RNR regulation and dNTP pools in S. cerevisiae. (A) Schematic representation of yeast RNR. The large subunit is an Rnr1 dimer, the small subunit is an Rnr2/Rnr4 heterodimer, although higher order complexes may also exist. Rnr3 (not shown) is a non-essential DNA-damage-inducible Rnr1 paralogue, which can form Rnr3 homodimers or heterodimers with Rnr1. The specificity effectors contact both loop 1 (blue) and loop 2 (green), while the substrates contact only loop 2. The specificity information is transmitted to the catalytic sites (red) via loop 2. (B) The approximate number of dNTP molecules present at any given moment in S-phase (S) and the total number of dNTP molecules required for the duplication of the yeast nuclear DNA (T). Numbers above the bars indicate the S/T ratios in percent.

Figure 2.

Overexpression of loop 2 rnr1 mutants leads to imbalanced dNTP pools. (A) Sequence alignment of loop 2 in RNRs from different organisms. Numbering is according to yeast Rnr1. The residues mutated in this study are boxed. (B) Incubation of yeast strains harbouring the indicated plasmids in SC – URA + GAL media for 3 h results in overexpression of Rnr1 proteins. (C) dNTP pools in the wild-type strain grown in SC + GLU media (28 pmol dCTP, 62 pmol dTTP, 51 pmol dATP and 15 pmol dGTP per 10⁸ cells) and mutant strains grown in SC – URA + GAL media for at least six generations. Two independent isogenic strains for each genotype were analysed. Data are represented as mean ± SD. Numbers above the bars indicate fold-increase (green) or fold-decrease (red) of the dNTP concentration relative to wild-type.

Figure 3.

Effects of dNTP pool imbalances on cell cycle and proliferation. (A) Representative HPLC chromatograms demonstrating the OD₂₆₀ absorbance of dNTPs in the wild-type (green), rnr1-Q288A (red) and rnr1-Y285A (blue) strains. Some of the dNTP peaks are invisible at this level of magnification. (B) dNTP pools in the wild-type (41 pmol dCTP, 81 pmol dTTP, 35 pmol dATP and 15 pmol dGTP per 10⁸ cells) and mutant strains with the integrated rnr1 alleles grown in YPAD media. Two independent isogenic strains for each genotype were analysed. Data are represented as mean ± SD. Numbers above the bars indicate fold-increase (green) or fold-decrease (red) of the dNTP concentration relative to wild-type. (C) Flow cytometry histograms for the strains indicated in (B). Peaks with less than G₁ DNA content (prominent in rnr1-Q288A and rnr1-R293A and indicated by brackets) represent dead cells and cell debris. (D) YPAD plates with strains indicated in (B). (E) Proliferation curves. Two independent isogenic strains for each genotype were grown overnight in YPAD, re-inoculated into fresh YPAD at the OD₆₀₀ = 0.1, the OD₆₀₀ was measured every hour and the values were plotted on log₂ scale. Data are represented as mean ± SD. The slow growing rnr1-Q288A and rnr1-R293A did not show any lag phase as their overnight cultures did not reach the stationary phase. (F) Tetrad analysis demonstrating that rnr1-293A relies on RNR3 for survival.

Figure 4.

The S-phase checkpoint is not activated in rnr1-Y285F and rnr1-Y285A strains. (A) Rnr3 and Rnr4 proteins are indicators of the S-phase checkpoint activation. Western blot analysis demonstrating that the increase of Rnr3 and Rnr4 levels in response to DNA damage by methyl methane sulphonate (MMS) depends on a functional S-phase checkpoint (presence of RAD53). (B) Western blot analysis demonstrating that the Rnr2, Rnr3 and Rnr4 levels are not elevated in rnr1-Y285F and rnr1-Y285A, whereas the Rnr2, Rnr3 and Rnr4 levels are elevated in rnr1-Q288A and rnr1-R293A to the similar level as after treatment by DNA damaging [4-nitroquinoline oxide (4-NQO) and MMS] and replication blocking [hydroxyurea (HU)] agents. (C) Tetrad analysis demonstrating that the rnr1-R293A strain requires DUN1 for survival.