Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase.

Abstract

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 subnnit of ribonucleotide reductase, RNR1 and RNR3. They share -80% amino acid identity with each other and 60% with the mammalian homolog, MI. 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 rarl 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 POLl mRNA, being expressed in the late Gz and S phases of the cell cycle. Progression from the a-factor-induced G~ block to induction of RNR1 mRNA is blocked by cycloheximide, further defining the requirement for protein synthesis in the G~-to S-phase transition. Both RNR1 and RNR3 transcripts are inducible by treatments that damage DNA, such as 4-nitroquinoline-l-oxide and methylmethanesulfonate, or block DNA replication, such as hydroxyurea. RNR1 is inducible 3-to 5-fold, and RNR3 is inducible > 100-fold. When MATa ceils are arrested in GI by a-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.

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

“…This organism possesses no deoxynucleoside kinase activities, and thus, dNTP synthesis is entirely dependent on RNR (72). In S. cerevisiae, the levels of the subunits are controlled transcriptionally and the level of mRNA of the α subunit is increased during the S-phase of the cell cycle during normal growth, while the levels of mRNA for the β and β′ subunits remain largely unchanged (22,25,27,31). We have recently shown that the mRNA levels are correlated with the protein levels (D. L. Perlstein, M. Huang, and J. Stubbe, unpublished results).…”

RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae.

“…6B). Because rad9 mutants show normal signaling for HU-induced RNR3 expression and are not themselves sensitive to HU (Elledge and Davis 1990), it was anticipated that rad9 mutants would have no effect on HUsensitivity of po12 mutants. Surprisingly, rad9 has a pronounced effect indicating that RAD9 may have a minor role in the response to HU arrest during S phase that is not detectable until the major pathway is knocked out.…”

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

“…Yeast ribonucleotide reductase, a multi-protein complex, catalyzes one step in the dNTP production pathway. It is activated both as a normal part of the cell cycle and also in response to DNA damage [21,22,23,24,25]. RNR1 mRNA levels vary considerably with the cell cycle; the peak level occurs in the G1 phase [26].…”