No abstract

Inhibition of DNA synthesis prevents mitotic entry through the action of the S-phase checkpoint. We have isolated S-phase arrest-defective {sad) mutants that show lethality in the presence of the DNA synthesis inhibitor hydroxyurea (HU). Several of these mutants show phenotypes consistent with inappropriate mitotic entry in the presence of unreplicated DNA, indicating a defect in the S-phase checkpoint, sadl mutants are additionally defective for the G^ and Gj DNA damage checkpoints, and for DNA damage-induced transcription of RNR2 and RNR3. The transcriptional response to DNA damage requires activation of the Dunl protein kinase. Activation of Dunl in response to replication blocks or DNA damage is blocked in sadl mutants. The HU sensitivity of sadl mutants is suppressed by mutations in CKSl, a subunit of the p34^"*^^* kinase, further establishing a link between cell cycle progression and lethality, sadl mutants are allelic to rad53, a radiation-sensitive mutant. SADl encodes an essential protein kinase. The observation that SADl controls three distinct checkpoints suggests a common mechanism for cell cycle arrest at these points. Together, these observations implicate protein phosphorylation in the cellular response to DNA damage and replication blocks. A successful eukaryotic cell division requires that cer tain cellular processes occur in a defined order and are coupled so that the initiation of one event is dependent on the completion of another. In most cell types, entry into mitosis is dependent on the completion of DNA synthesis. Cells blocked for DNA replication arrest in S phase and delay mitotic initiation. Cell cycle arrest in response to S-phase inhibition is attributable to the pres ence of a feedback control or checkpoint mechanism (for reviews on cell cycle checkpoints, see Hartwell and

We have constructed a strain of Saccharomyces cerevisiae with a deletion of the YKL510 open reading frame, which was initially identified in chromosome XI as a homolog of the RAD2 nucleotide excision repair gene (A. Jacquier, P. Legrain, and B. Dujon, Yeast 8:121-132, 1992). The mutant strain exhibits increased sensitivity to UV light and to the alkylating agent methylmethane sulfonate but not to ionizing radiation. We have renamed the YKL510 open reading frame the RAD27 gene, in keeping with the accepted nomenclature for radiationsensitive yeast mutants. Epistasis analysis indicates that the gene is in the RAD6 group of genes, which are involved in DNA damage tolerance. The mutant strain also exhibits increased plasmid loss, increased spontaneous mutagenesis, and a temperature-sensitive lethality whose phenotype suggests a defect in DNA replication. Levels of the RAD27 gene transcript are cell cycle regulated in a manner similar to those for several other genes whose products are known to be involved in DNA replication. We discuss the possible role of Rad27 protein in DNA repair and replication.

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-l-oxide (4NQO), or yradiation after release from G, arrest induced by a factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage (Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-3221, the normal execution of DNA damageinduced G, arrest depends on a functional yeast RAD9 gene.We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G, arrest point but prior to replicative DNA synthesis, S phaseassociated maximal induction of histone H2A mRNA, and bud emergence. Additional support for the notion of regulated checkpoints derives from several recent observations. A decrease in the fraction of S phase cells and an increase in the fraction of G, cells have been correlated with an increase in the level of p53 protein (11) in several mammalian cell lines following exposure to y irradiation. Furthermore, p53 mutant cells failed to arrest in G1 after y irradiation (12,13). An increase in p53 levels was not observed in irradiated AT cells (13). Hence, p53 and the AT gene(s) may participate in a signal transduction pathway that regulates cell cycle arrest after DNA damage.In S. cerevisiae nutrient deprivation or exposure to mating pheromone (a factor) results in the arrest of haploid cells in G1. This arrest is associated with a failure to activate the CDC28-encoded protein kinase, a homologue ofthe Cdc2 and p34 proteins in Schizosaccharomyces pombe and mammalian cells, respectively. In S. cerevisiae reentry to the cell cycle depends on a transition from this restriction point, termed START (14,15).In the present study we have investigated the effect of DNA damage on the progression of yeast cells through the cell cycle. We show that exposure of synchronized cells to UV radiation, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or 'y radiation results in G1 arrest. We additionally show that this arrest requires a functional RAD9 gene. The dependence of G1 arrest on a gene previously implicated in arrest in the G2 phase (5) suggests that arrest during G1 is a regulated phenomenon that operates as a cell cycle checkpoint in yeast cells exposed to various types of DNA damage. MATERIALS AND METHODS

How mitochondrial DNA (mtDNA) copy number is determined and modulated according to cellular demands is largely unknown. Our previous investigations of the related DNA helicases Pif1p and Rrm3p uncovered a role for these factors and the conserved Mec1/Rad53 nuclear checkpoint pathway in mtDNA mutagenesis and stability in Saccharomyces cerevisiae. Here, we demonstrate another novel function of this pathway in the regulation of mtDNA copy number. Deletion of RRM3 or SML1, or overexpression of RNR1, which recapitulates Mec1/Rad53 pathway activation, resulted in an approximately twofold increase in mtDNA content relative to the corresponding wild-type yeast strains. In addition, deletion of RRM3 or SML1 fully rescued the ϳ50% depletion of mtDNA observed in a pif1 null strain. Furthermore, deletion of SML1 was shown to be epistatic to both a rad53 and an rrm3 null mutation, placing these three genes in the same genetic pathway of mtDNA copy number regulation. Finally, increased mtDNA copy number via the Mec1/Rad53 pathway could occur independently of Abf2p, an mtDNA-binding protein that, like its metazoan homologues, is implicated in mtDNA copy number control. Together, these results indicate that signaling through the Mec1/Rad53 pathway increases mtDNA copy number by altering deoxyribonucleoside triphosphate pools through the activity of ribonucleotide reductase. This comprises the first linkage of a conserved signaling pathway to the regulation of mitochondrial genome copy number and suggests that homologous pathways in humans may likewise regulate mtDNA content under physiological conditions. INTRODUCTIONSince the discovery that the mitochondrial genome is present at multiple copies per cell (100 -10,000 in humans) and is subject to dynamic regulation with regard to tissue type, metabolic signals, and environmental stimuli, an understanding of the pathways and mechanisms that regulate cellular mitochondrial DNA (mtDNA) copy number has been sought (Moraes, 2001). Although nuclear gene products that have direct roles in mtDNA replication or stability have been implicated in copy number regulation (Schultz et al., 1998;Zelenaya-Troitskaya et al., 1998;Ekstrand et al., 2004;Matsushima et al., 2004;Tyynismaa et al., 2004), signaling pathways involved in modulating cellular mtDNA content have not been fully elucidated. However, important insight into mtDNA copy number regulation and its clinical significance has been gleaned from the study of human mitochondrial disease patients. For example, several mtDNA-depletion syndromes have been characterized, the hallmark of which is decreased mtDNA copy number and/or integrity in certain tissues (Moraes et al., 1991;Elpeleg et al., 2002). Identification of the nuclear genetic defects underlying several of these diseases has underscored a critical role for cellular deoxyribonucleoside triphosphate (dNTP) pools in mtDNA copy number and stability. For example, mutations in the mitochondrial thymidine kinase gene cause mitochondrial depletion myopathy (Saada et al., 2001), mutations...