In eukaryotes, DNA damage elicits a multifaceted response that includes cell cycle arrest, transcriptional activation of DNA repair genes, and, in multicellular organisms, apoptosis. We demonstrate that in Saccharomyces cerevisiae, DNA damage leads to a 6- to 8-fold increase in dNTP levels. This increase is conferred by an unusual, relaxed dATP feedback inhibition of ribonucleotide reductase (RNR). Complete elimination of dATP feedback inhibition by mutation of the allosteric activity site in RNR results in 1.6-2 times higher dNTP pools under normal growth conditions, and the pools increase an additional 11- to 17-fold during DNA damage. The increase in dNTP pools dramatically improves survival following DNA damage, but at the same time leads to higher mutation rates. We propose that increased survival and mutation rates result from more efficient translesion DNA synthesis at elevated dNTP concentrations.

The evolutionarily conserved protein kinases Mec1 and Rad53 are required for checkpoint response and growth. Here we show that their role in growth is to remove the ribonucleotide reductase inhibitor Sml1 to ensure DNA replication. Sml1 protein levels¯uctuate during the cell cycle, being lowest during S phase. The disappearance of Sml1 protein in S phase is due to post-transcriptional regulation and is associated with protein phosphorylation. Both phosphorylation and diminution of Sml1 require MEC1 and RAD53. Moreover, failure to remove Sml1 in mec1 and rad53 mutants results in incomplete DNA replication, defective mitochondrial DNA propagation, decreased dNTP levels and cell death. Interestingly, similar regulation of Sml1 also occurs after DNA damage. In this case, the regulation requires MEC1 and RAD53, as well as other checkpoint genes. Therefore, Sml1 is a new target of the DNA damage checkpoint and its removal is a conserved function of Mec1 and Rad53 during growth and after damage. Keywords: checkpoint/Mec1/protein phosphorylation/ Rad53/ribonucleotide reductase IntroductionIn the yeast Saccharomyces cerevisiae, Mec1 and Rad53 protein kinases are essential both after DNA damage and during cell growth (Zheng et al., 1993;Kato and Ogawa, 1994;Weinert et al., 1994). In response to DNA damage, they function as signal transducers in all known checkpoint pathways (reviewed in Elledge, 1996). Rad53 usually functions downstream of Mec1 and, together, they receive signals from upstream sensor proteins transmitting them to components of the cell cycle engine. Consequently, cell cycle progression is arrested or delayed, providing time for repair. In addition, Mec1 and Rad53 also increase the capacity of the cell to repair DNA lesions. One route is by the transcriptional induction of various DNA repair proteins, including ribonucleotide reductase (RNR), the enzyme that catalyzes the ratelimiting step of both deoxyribonucleotide (dNTP) and DNA synthesis (reviewed in Reichard, 1988). However, additional interfaces between the Mec1/Rad53 checkpoint pathway and DNA repair are probably required to maximize protection of genetic integrity. A better understanding of such interactions relies on the discovery of new targets of checkpoint control.The checkpoint function of Mec1 and Rad53 is evolutionarily conserved. Their mammalian homologs, ATM/ATR and CHK2, also function as signal transducers and affect multiple components of the cell cycle and DNA repair machinery during the response to DNA damage (reviewed in Rotman and Shiloh, 1999). For example, ATM/ATR and CHK2 activate and stabilize p53, which in turn leads to the transcriptional induction of a variety of genes, including that of RNR (Tanaka et al., 2000;Zhao et al., 2000a; reviewed in Caspari, 2000).The conservation between Mec1/Rad53 and their mammalian homologs may extend beyond their checkpoint functions. These proteins are also important for normal cell growth; in yeast, deletion of MEC1 or RAD53 is lethal (Zheng et al., 1993;Kato and Ogawa, 1994) and, in mice, de...

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides; this step is rate-limiting in DNA precursor synthesis. A number of regulatory mechanisms ensure optimal deoxyribonucleotide pools, which are essential for cell viability. The best studied mechanisms are transcriptional regulation of the RNR genes during the cell cycle and in the response to DNA damage, and the allosteric regulation of ribonucleotide reductase by nucleoside triphosphates. Recently, another mode of RNR regulation has been hypothesized in yeast. A novel protein, Sml1, was shown to bind to the Rnr1 protein of the yeast ribonucleotide reductase; this interaction was proposed to inhibit ribonucleotide reductase activity when DNA synthesis is not required (Zhao, X., Muller, E.G.D., and Rothstein, R. (1998) Mol. Cell 2, 329 -340). Here, we use highly purified recombinant proteins to directly demonstrate that the Sml1 protein is a strong inhibitor of yeast RNR. The Sml1p specifically binds to the yeast Rnr1p in a 1:1 ratio with a dissociation constant of 0.4 M. Interestingly, Sml1p also specifically binds to the mouse ribonucleotide reductase R1 protein. However, the inhibition observed in an in vitro mouse ribonucleotide reductase assay is less pronounced than the inhibition in yeast and probably occurs via a different mechanism.Ribonucleotide reductase (RNR) 1 plays a crucial role in DNA synthesis, by catalyzing the direct reduction of all four ribonucleotides to deoxyribonucleotides. Both the yeast and mammalian ribonucleotide reductases belong to the ribonucleotide reductase class Ia; in this class, the active enzymes consist of a large subunit and a small one (1). In mammalian cells, these two non-identical homodimeric subunits are encoded by the R1 and R2 genes, respectively. The R1 protein contains redoxactive dithiols, the active site binding nucleoside diphosphate substrates and binding sites for nucleoside triphosphates that act as allosteric effectors. Substrate specificity is controlled by binding of ATP, dATP, dTTP, or dGTP to a specificity site, whereas overall activity is controlled by binding of ATP (active) or dATP (inactive) to an activity site (2). Failure to control the size of dNTP pools and/or their relative amounts leads to cell death or genetic abnormalities (3).The R2 protein contributes a tyrosyl-free radical, which is essential for RNR activity; this radical is generated by a binuclear iron center (2). The flexible C-terminal tail of the R2 polypeptide chain is essential for the R1 and R2 interaction, and upon binding to the R1 protein, the flexible R2 protein tail becomes rigid (4). Peptides and peptidomimetics corresponding to the R2 protein C-terminal inhibit ribonucleotide reductase in a species specific way (5).In Saccharomyces cerevisiae, there are two genes encoding R1-like proteins, RNR1 and RNR3 (6); there are also two genes encoding R2-like proteins, RNR2 (7,8) and RNR4 (9, 10). The yeast ribonucleotide reductase genes are one of the targets of the Mec1-Rad53-dependent...

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Eukaryotes have an ␣2␤2 form of RNR consisting of two homodimeric subunits, proteins R1 (␣2) and R2 (␤2). The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors. The R2 protein is a radical storage device containing an iron centergenerated tyrosyl free radical. Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding. Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p (␤) is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p͞Rnr4p complex. This complex sediments at 5.6 S as a ␤␤ heterodimer in a sucrose gradient. In the presence of Rnr1p, both polypeptides of the Rnr2p͞Rnr4p heterodimer cosediment at 9.7 S as expected for an ␣2␤␤ heterotetramer, where Rnr4p plays an important role in the interaction between the ␣2 and the ␤␤ subunits. The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity. This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy. Taken together, our results show that the Rnr2p͞Rnr4p heterodimer is the active form of the yeast RNR small subunit.