title: Ixr1 is required for the expression of the ribonucleotide reductase Rnr1 and maintenance of dNTP pools.
abstract: The Saccharomyces cerevisiae Dun1 protein kinase is a downstream target of the conserved Mec1-Rad53 checkpoint pathway. Dun1 regulates dNTP pools during an unperturbed cell cycle and after DNA damage by modulating the activity of ribonucleotide reductase (RNR) by multiple mechanisms, including phosphorylation of RNR inhibitors Sml1 and Dif1. Dun1 also activates DNA-damage-inducible genes by inhibiting the Crt1 transcriptional repressor. Among the genes repressed by Crt1 are three out of four RNR genes: RNR2, RNR3, and RNR4. The fourth RNR gene, RNR1, is also DNA damage-inducible, but is not controlled by Crt1. It has been shown that the deletion of DUN1 is synthetic lethal with the deletion of IXR1, encoding an HMG-box-containing DNA binding protein, but the reason for this lethality is not known. Here we demonstrate that the dun1 ixr1 synthetic lethality is caused by an inadequate RNR activity. The deletion of IXR1 results in decreased dNTP levels due to a reduced RNR1 expression. The ixr1 single mutants compensate for the reduced Rnr1 levels by the Mec1-Rad53-Dun1-Crt1-dependent elevation of Rnr3 and Rnr4 levels and downregulation of Sml1 levels, explaining why DUN1 is indispensible in ixr1 mutants. The dun1 ixr1 synthetic lethality is rescued by an artificial elevation of the dNTP pools. We show that Ixr1 is phosphorylated at several residues and that Ser366, a residue important for the interaction of HMG boxes with DNA, is required for Ixr1 phosphorylation. Ixr1 interacts with DNA at multiple loci, including the RNR1 promoter. Ixr1 levels are decreased in Rad53-deficient cells, which are known to have excessive histone levels. A reduction of the histone gene dosage in the rad53 mutant restores Ixr1 levels. Our results demonstrate that Ixr1, but not Dun1, is required for the proper RNR1 expression both during an unperturbed cell cycle and after DNA damage.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Olga
authors.initials: O
authors.lastname: Tsaponina
authors.forename: Emad
authors.initials: E
authors.lastname: Barsoum
authors.forename: Stefan U
authors.initials: SU
authors.lastname: Aström
authors.forename: Andrei
authors.initials: A
authors.lastname: Chabes
mesh_terms: D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D006609:High Mobility Group Proteins / Q000235:genetics* / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D015363:Quinolones / Q000494:pharmacology; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D014158:Transcription, Genetic
chemical_list: C120195:4-nitroquinolone-1-oxide; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D006657:Histones; C082364:IXR1 protein, S cerevisiae; D019384:Nucleic Acid Synthesis Inhibitors; D015363:Quinolones; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015112:4-Nitroquinoline-1-oxide; D012264:Ribonucleotide Reductases; C551024:Rnr1 protein, S cerevisiae; C517042:RNR3 protein, S cerevisiae; D012262:Ribonucleoside Diphosphate Reductase; C523202:Rnr4 protein, S cerevisiae; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D006918:Hydroxyurea