Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae

Abstract

In eukaryotic cells the concentration of dNTP is highest in S phase and lowest in G1 phase and is controlled by ribonucleotide reductase (RNR). RNR activity is eliminated in all eukaryotes in G1 phase by a variety of mechanisms: transcriptional regulation, small inhibitory proteins, and protein degradation. After activation of RNR upon commitment to S phase, dATP feedback inhibition ensures that the dNTP concentration does not exceed a certain maximal level. It is not apparent why limitation of dNTP concentration is necessary in G1 phase. In principle, dATP feedback inhibition should be sufficient to couple dNTP production to utilization. We demonstrate that in Saccharomyces cerevisiae constitutively high dNTP concentration transiently arrests cell cycle progression in late G1 phase, affects activation of origins of replication, and inhibits the DNA damage checkpoint. We propose that fluctuation of dNTP concentration controls cell cycle progression and the initiation of DNA replication.

Fig. 1.

Expression of RNR1 alleles increases [dNTP] and decreases [NTP] but does not affect transcription. (A) Changes in [dNTP] in pGAL-RNR1 and pGAL-rnr1-D57N strains after a 3-h gal induction. [dNTP] in the wild-type W1588-4C strain was assigned a value of 1. (B) [NTP] in pGAL-RNR1 (AC438) and pGAL-rnr1-D57N (AC439) strains after a 3-h gal induction, in percentage of [NTP] in a wild-type strain. (C–F) pGAL (AC437), pGAL-RNR1 (AC438), and pGAL-rnr1-D57N (AC439) strains were grown in minimal media without methionine until an OD₆₀₀ of 0.2 and after the addition of 2% gal or raf incubated for 4 h. (C) Flow-cytometric analysis of pGAL (Left), pGAL-RNR1 (Center), and pGAL-rnr1-D57N(Right). (D) Ethidium bromide staining showing equal amounts of 25S and 18S rRNA. (E and F) Ethidium bromide staining (E) and corresponding autoradiography (F) of the [³H]methionine-labeled rRNA sample from the pGAL-rnr1-D57N (AC439) strain. + and − indicate presence and absence of gal.

Fig. 2.

Expression of pGAL-rnr1-D57N leads to DNA damage sensitivity and defects in DNA damage checkpoint. (A) Overnight cultures of pGAL (AC437), pGAL-RNR1 (AC438), and pGAL-rnr1-D57N (AC439) strains grown in YPD were spotted at 10-fold serial dilutions on YPD, YPGal, and YPGal/0.3 mg/4-nitroquinoline 1-oxide plates. (B) pGAL-rnr1-D57N (AC439) strain was grown in YPRaf to an OD₆₀₀ of 0.2, and half of the culture was induced by 2% gal for 2 h (indicated by +). After that, 0.01% MMS was added (indicated by +), and samples were collected for analysis by Western blotting (Upper) and SDS/PAGE (Lower) at 45 and 90 min. (C) pGAL-rnr1-D57N (AC439) strain was grown in YPRaf to an OD₆₀₀ of 0.2, and half of the culture was induced by 2% gal for 2 h (indicated by GAL). After that, 0.01% MMS was added (indicated by +MMS), and samples were collected for flow-cytometric analysis.

Fig. 3.

Expression of pGAL-rnr1-D57N does not activate Rad53 and does not induce transcription of RNR3. (A) pGAL (AC437) and pGAL-rnr1-D57N (AC439) strains were grown in YPRaf to an OD₆₀₀ of 0.2, and, after the addition of either 0.01% MMS, 2% gal, or no addition of drug, they were incubated for 3 h. The expression of Rnr1-D57N was analyzed by SDS/PAGE (Upper) and Rad53 phosphorylation by Western blotting (Lower). M, protein marker lanes. (B) pGAL-rnr1-D57N RNR3-HA (AC454-4F) strain was grown in liquid YPRaf to an OD₆₀₀ of 0.2, and, after the addition of either 0.3 mg of 4-nitroquinoline 1-oxide, 2% gal, or no addition of drug, they were incubated for 3 h. The expression of Rnr1-D57N was analyzed by SDS/PAGE (Upper), and the expression of Rnr3-HA was analyzed by Western blotting (Lower).

Fig. 4.

Loading of Cdc45 onto chromatin is inhibited by expression of pGAL-rnr1-D57N. (A) pGAL-rnr1-D57N (AC439) strain was treated as shown in the outline. The samples were collected at 10-min intervals for analysis of Cdc45 and Mcm2 association with chromatin and for flow-cytometric analysis. (B) The pGAL-rnr1-D57N dbf2-1 (AC473) strain was treated as shown in the outline. The samples were collected at 10-min intervals for analysis of Cdc45, Mcm2, Orc3, and Orc5 association with chromatin and for flow-cytometric analysis.

Fig. 5.

Elevation of [dNTP] results in synthetic sickness or lethality in the orc2-1 and orc5-1 mutants. (A) Wild-type (AC476-1D), orc2-1 (AC476-1A), orc2-1 sml1 pGAL-RNR1 (AC476-5B), and sml1 pGAL-RNR1 (AC475-4I) strains were streaked on YPGal and incubated at 24°C. (Right) A magnification showing synthetic sickness of orc2-1 plus pGAL-RNR1 (slower growth, uneven colonies). (B) orc5-1 (AC474-3H), orc5-1 sml1 pGAL-RNR1 (AC474-3G), and sml1 mec1 pGAL-RNR1 (AC474-3F) strains were streaked on YPGal and incubated at 33°C. (C) Wild-type (AC459-1A), orc5-1 pGAL-rnr1-D57N (AC459-1D), orc5-1(AC459-1C), and pGAL-rnr1-D57N (AC459-1B) strains were streaked on YPGal and incubated at 33°C.