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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) provides the cell with a balanced supply of deoxyribonucleoside triphosphates (dNTP) for DNA synthesis. In budding yeast DNA damage leads to an up-regulation of RNR activity and an increase in dNTP pools, which are essential for survival. Mammalian cells contain three non-identical subunits of RNR; that is, one homodimeric large subunit, R1, carrying the catalytic site and two variants of the homodimeric small subunit, R2 and the p53-inducible p53R2, each containing a tyrosyl free radical essential for catalysis. S-phase-specific DNA replication is supported by an RNR consisting of the R1 and R2 subunits. In contrast, DNA damage induces expression of the R1 and the p53R2 subunits. We now show that neither logarithmically growing nor G o /G 1 -synchronized mammalian cells show any major increase in their dNTP pools after DNA damage. However, non-dividing fibroblasts expressing the p53R2 protein, but not the R2 protein, have reduced dNTP levels if exposed to the RNR-specific inhibitor hydroxyurea, strongly indicating that there is ribonucleotide reduction in resting cells. The slow, 4-fold increase in p53R2 protein expression after DNA damage results in a less than 2-fold increase in the dNTP pools in G o /G 1 cells, where the pools are about 5% that of the size of the pools in S-phase cells. Our results emphasize the importance of the low constitutive levels of p53R2 in mammalian cells, which together with low levels of R1 protein may be essential for the supply of dNTPs for basal levels of DNA repair and mitochondrial DNA synthesis in G o /G 1 cells. Mammalian cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs)2 for DNA replication and repair. The rate-limiting step in the formation of DNA precursors is the de novo reduction of ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates by the enzyme ribonucleotide reductase (RNR) (1). In S phase, the mammalian RNR enzyme is composed of the homodimeric R1 and R2 subunits, which together form a heterotetrameric active enzyme. The large R1 protein (90 kDa) carries the active site, whereas the small R2 protein (45 kDa) contains a diferric iron center generating a tyrosyl free radical necessary for catalysis (1, 2). An additional mammalian RNR protein, p53R2, was identified in 2000 (3, 4). Like the homologous R2 protein, p53R2 contains a tyrosyl free radical and forms an active RNR complex with the R1 protein in vitro (5). The tyrosyl free radical of both the R2 and the p53R2 proteins is specifically destroyed by the RNR inhibitor hydroxyurea (5, 6).Because unbalanced dNTP pools can cause genetic abnormalities and cell death (1), RNR activity is tightly regulated in mammalian cells by S-phase-specific transcription of the R1 and R2 genes (7-9), binding of nucleoside triphosphate allosteric effectors to the R1 protein (10), and anaphase promoting complex-Cdh1-mediated degradation of the R2 protein in late mitosis (11,12). In cycling cells, the S-phase-dependent activity of the RNR complex is l...

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