The POL1 gene from the fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha

Summary The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 by intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.     

DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion

Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA^Gln and tRNA^Glu respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S‐phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate‐limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage.     

Increased tRNA modification and gene-specific codon usage regulate cell cycle progression during the DNA damage response

S-phase and DNA damage promote increased ribonucleotide reductase (RNR) activity. Translation of RNR1 has been linked to the wobble uridine modifying enzyme tRNA methyltransferase 9 (Trm9). We predicted that changes in tRNA modification would translationally regulate RNR1 after DNA damage to promote cell cycle progression. In support, we demonstrate that the Trm9-dependent tRNA modification 5-methoxycarbonylmethyluridine (mcm?U) is increased in hydroxyurea (HU)-induced S-phase cells, relative to G? and G?, and that mcm?U is one of 16 tRNA modifications whose levels oscillate during the cell cycle. Codon-reporter data matches the mcm?U increase to Trm9 and the efficient translation of AGA codons and RNR1. Further, we show that in trm9Δ cells reduced Rnr1 protein levels cause delayed transition into S-phase after damage. Codon re-engineering of RNR1 increased the number of trm9Δ cells that have transitioned into S-phase 1 h after DNA damage and that have increased Rnr1 protein levels, similar to that of wild-type cells expressing native RNR1. Our data supports a model in which codon usage and tRNA modification are regulatory components of the DNA damage response, with both playing vital roles in cell cycle progression.     

Giant yeast cells with nonrecyclable ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and thereby provides the precursors required for DNA synthesis and repair. In an attempt to test cell resistance to a permanent replicational stress, we constructed a mutant Saccharomyces cerevisiae strain containing exclusively nonrecyclable catalytic subunits of RNR that become inactivated following the reduction of one ribonucleoside diphosphate. In this rnr1C883A rnr3Δ mutant, the synthesis of each deoxyribonucleotide thus requires the production of one Rnr1C883A protein, which means that 26 million Rnr1C883A proteins (half the protein complement of a wild-type cell) have to be produced during each cell cycle. rnr1C883A rnr3Δ cells grow under constant replicational stress, as evidenced by the constitutive activation of the checkpoint effector Rad53, and their S phase is considerably extended compared to the wild type. rnr1C883A rnr3Δ mutants also display additional abnormalities such as a median cell volume increased by a factor of 8, and the presence of massive inclusion bodies. However, they exhibit a good plating efficiency and can be propagated indefinitely. rnr1C883A rnr3Δ cells, which can be used as a protein overexpression system, thus illustrate the robustness of S. cerevisiae to multiple physiological parameters.     

Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint

A balanced supply of deoxyribonucleoside triphosphates (dNTPs) is one of the key prerequisites for faithful genome duplication. Both the overall concentration and the balance among the individual dNTPs (dATP, dTTP, dGTP, and dCTP) are tightly regulated, primarily by the enzyme ribonucleotide reductase (RNR). We asked whether dNTP pool imbalances interfere with cell cycle progression and are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. By introducing single amino acid substitutions in loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR, we obtained a collection of strains with various dNTP pool imbalances. Even mild dNTP pool imbalances were mutagenic, but the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity. The S-phase checkpoint was activated by the depletion of one or several dNTPs. In contrast, when none of the dNTPs was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression.     

Kin3 protein,a NIMA-related kinase of Saccharomyces cerevisiae,is involved in DNA adduct damage response

Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.     

Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability.

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.     

DNA replication stress-induced loss of reproductive capacity in S. cerevisiae and its inhibition by caloric restriction

In many organisms, attenuation of growth signaling by caloric restriction or mutational inactivation of growth signaling pathways extends lifespan and protects against cancer and other age-related diseases. The focus of many efforts to understand these effects has been on the induction of oxidative stress defenses that inhibit cellular senescence and cell death. Here we show that in the model organism S. cerevisiae, growth signaling induces entry of cells in stationary phase into S phase in parallel with loss of reproductive capacity, which is enhanced by elevated concentrations of glucose. Overexpression of RNR1 encoding a ribonucleotide reductase subunit required for the synthesis of deoxynucleotide triphosphates and DNA replication suppresses the accelerated loss of reproductive capacity of cells cultured in high glucose. The reduced reproductive capacity of these cells is also suppressed by excess threonine, which buffers dNTP pools when ribonucleotide reductase activity is limiting. Caloric restriction or inactivation of the AKT homolog Sch9p inhibits senescence and death in stationary phase cells caused by the DNA replication inhibitor hydroxyurea or by inactivation of the DNA replication and repair proteins Sgs1p or Rad27p. Inhibition of DNA replication stress represents a novel mechanism by which caloric restriction promotes longevity in S. cerevisiae. A similar mechanism may promote longevity and inhibit cancer and other age-related diseases in humans.     

α‐Synuclein toxicity in yeast and human cells is caused by cell cycle re‐entry and autophagy degradation of ribonucleotide reductase 1

α‐Synuclein (aSyn) toxicity is associated with cell cycle alterations, activation of DNA damage responses (DDR), and deregulation of autophagy. However, the relationships between these phenomena remain largely unknown. Here, we demonstrate that in a yeast model of aSyn toxicity and aging, aSyn expression induces Ras2‐dependent growth signaling, cell cycle re‐entry, DDR activation, autophagy, and autophagic degradation of ribonucleotide reductase 1 (Rnr1), a protein required for the activity of ribonucleotide reductase and dNTP synthesis. These events lead to cell death and aging, which are abrogated by deleting RAS2, inhibiting DDR or autophagy, or overexpressing RNR1. aSyn expression in human H4 neuroglioma cells also induces cell cycle re‐entry and S‐phase arrest, autophagy, and degradation of RRM1, the human homologue of RNR1, and inhibiting autophagic degradation of RRM1 rescues cells from cell death. Our findings represent a model for aSyn toxicity that has important implications for understanding synucleinopathies and other age‐related neurodegenerative diseases.     

Dissecting the DNA damage response using functional genomics approaches in S. cerevisiae

The faithful transmission of genetic information from a mother to daughter cells can only occur if the integrity of the genome is preserved. DNA transactions within cells are tightly regulated to prevent DNA damage. When events that challenge genome integrity do occur, a complex web of DNA damage response pathways comes into play. Studies in model organisms have contributed significantly to the understanding of these pathways. In the last decade, the development of functional genomics techniques in S.cerevisiae has ushered in systematic approaches for the study of complex cellular processes. These methods have enabled the systematic interrogation of the DNA damage response.     

Identification of the DNA damage-responsive elements of therhp51+ gene, arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51 ⁺ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51 ⁺ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51 ⁺ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51 ⁺ expression.     

Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen

Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation.