Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase.
Ribonucleotide reductase activity is essential for progression through the cell cycle, catalyzing the rate-limiting step for the production of deoxyribonucleotides needed for DNA synthesis. The enzymatic activity of the enzyme fluctuates in the cell cycle with an activity maximum in S phase. We have identified and characterized two Saccharomyces cerevisiae genes encoding the regulatory subunit of ribonucleotide reductase, RNR1 and RNR3. They share approximately 80% amino acid identity with each other and 60% with the mammalian homolog, M1. Genetic disruption reveals that the RNR1 gene is essential for mitotic viability, whereas the RNR3 gene is not essential. A high-copy-number clone of RNR3 is able to suppress the lethality of rnr1 mutations. Analysis of mRNA levels in cell-cycle-synchronized cultures reveals that the RNR1 mRNA is tightly cell-cycle regulated, fluctuating 15- to 30-fold, and is coordinately regulated with the POL1 mRNA, being expressed in the late G1 and S phases of the cell cycle. Progression from the alpha-factor-induced G1 block to induction of RNR1 mRNA is blocked by cycloheximide, further defining the requirement for protein synthesis in the G1- to S-phase transition. Both RNR1 and RNR3 transcripts are inducible by treatments that damage DNA, such as 4-nitroquinoline-1-oxide and methylmethanesulfonate, or block DNA replication, such as hydroxyurea. RNR1 is inducible 3- to 5-fold, and RNR3 is inducible greater than 100-fold. When MATa cells are arrested in G1 by alpha-factor, RNR1 and RNR3 mRNA is still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. Inhibition of ribonucleotide reductase activity by hydroxyurea treatment results in arrest of the cell cycle in S phase as large budded, uninucleate cells. This specific cell-cycle arrest is independent of the RAD9 gene, defining a separate pathway for the coordination of DNA synthesis and cell-cycle progression.
Elledge
S J
SJ
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Davis
R W
RW
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D002453:Cell Cycle; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000096:biosynthesis; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D004271:DNA, Fungal; D005656:Fungal Proteins; D012264:Ribonucleotide Reductases