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  • 1.
    Barsoum, E.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Developmental Biology.
    Rajaei, Naghmeh
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Developmental Biology.
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Developmental Biology.
    RAS/Cyclic AMP and Transcription Factor Msn2 Regulate Mating and Mating-Type Switching in the Yeast Kluyveromyces lactis2011In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 10, no 11, p. 1545-1552Article in journal (Refereed)
    Abstract [en]

    In response to harsh environmental conditions, ascomycetes produce stress-resistant spores to promote survival. As sporulation requires a diploid DNA content, species with a haploid lifestyle, such as Kluyveromyces lactis, first induce mating in response to stress. In K. lactis, mating and mating-type switching are induced by the DNA-binding protein Mts1. Mts1 expression is known to be upregulated by nutrient limitation, but the mechanism is unknown. We show that a ras2 mutation results in a hyperswitching phenotype. In contrast, strains lacking the phosphodiesterase Pde2 had lower switching rates compared to that of the wild type (WT). As Ras2 promotes cyclic AMP (cAMP) production and Pde2 degrades cAMP, these data suggest that low cAMP levels induce switching. Because the MTS1 regulatory region contains several Msn2 binding sites and Msn2 is a transcription factor that is activated by low cAMP levels, we investigated if Msn2 regulates MTS1 transcription. Consistently with this idea, an msn2 mutant strain displayed lower switching rates than the WT strain. The transcription of MTS1 is highly induced in the ras2 mutant strain. In contrast, an msn2 ras2 double mutant strain displays WT levels of the MTS1 transcript, showing that Msn2 is a critical inducer of MTS1 transcription. Strains lacking Msn2 and Pde2 also exhibit mating defects that can be complemented by the ectopic expression of Mts1. Finally, we show that MTS1 is subjected to negative autoregulation, presumably adding robustness to the mating and switching responses. We suggest a model in which Ras2/cAMP/Msn2 mediates the stress-induced mating and mating-type switching responses in K. lactis.

  • 2.
    Barsoum, Emad
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Martinez, Paula
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    {alpha}3, a transposable element that promotes host sexual reproduction2010In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 24, no 15, p. 33-44Article in journal (Refereed)
    Abstract [en]

    Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

  • 3.
    Barsoum, Emad
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Sjöstrand, Jimmy O. O.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Ume6 is required for the MATa/MATα cellular identity and transcriptional silencing in Kluyveromyces lactis2010In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 184, no 4, p. 999-1011Article in journal (Refereed)
    Abstract [en]

    To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of the unscheduled meiotic gene expression 6 (UME6) gene in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Chromatin immunoprecipitation (ChIP) suggested that Ume6 acted directly by binding the cis-regulatory silencers of these loci. Unexpectedly, a MATa ume6 strain was mating proficient, whereas a MATα ume6 strain was sterile. This observation was explained by the fact that ume6 derepressed HMLα2 only weakly, but derepressed HMRa1 strongly. Consistently, two a/α-repressed genes (MTS1 and STE4) were repressed in the MATα ume6 strain, but were expressed in the MATa ume6 strain. Surprisingly, ume6 partially suppressed the mating defect of a MATa sir2 strain. MTS1 and STE4 were repressed in the MATa sir2 ume6 double-mutant strain, indicating that the suppression acted downstream of the a1/α2-repressor. We show that both STE12 and the MATa2/HMRa2 genes were overexpressed in the MATa sir2 ume6 strain. Consistent with the idea that this deregulation suppressed the mating defect, ectopic overexpression of Ste12 and a2 in a MATa sir2 strain resulted in efficient mating. In addition, Ume6 served as a block to polyploidy, since ume6/ume6 diploids mated as pseudo a-strains. Finally, Ume6 was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors.

  • 4.
    Barsoum, Emad
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Åström, Stefan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Regulation of mating type switching in Kluyveromyces lactis by the RAS/cAMP pathway and the transcription factor Msn2Manuscript (preprint) (Other academic)
    Abstract [en]

    We explored the regulation of mating type switching in Kluyveromyces lactis. Using an assay dependent on loss of a URA3 gene inserted into the MATa locus, we determined that the switching rate of a wild type strain grown in rich media was ~6X10-4 events/generation. In a genetic selection for identifying strains with increased switching rates, we found a strain with an insertion in the K. lactis RAS1 gene, encoding a small GTPase with a central role in growth regulation. Compromised Ras1 function leads to a lower cAMP level suggesting a role for cAMP in promoting switching. Consistent with this idea, a strain lacking the PDE2 gene, which encodes an enzyme that degrades cAMP, resulted in decreased switching rates. To explore how cAMP regulated switching, we investigated the transcription of the MTS1 gene, encoding an inducer of switching. The ras1 mutant strain contained 20-fold higher levels of the MTS1 mRNA compared to wild type, but in the pde2 mutant strain MTS1 transcription was repressed 5-fold. In addition, strains lacking the MSN2 gene, which encodes a transcription factor that binds the stress response element (STRE), expressed less MTS1 mRNA and had decreased switching rates. We suggest a model in which nutrient limitation induces switching through cAMP and Msn2-dependent transcriptional induction of the MTS1 gene.

  • 5.
    Bauer, Stefanie L.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Chen, Jiang
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Åström, Stefan U.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Helicase/SUMO-targeted ubiquitin ligase Uls1 interacts with the Holliday junction resolvase Yen12019In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 3, article id e0214102Article in journal (Refereed)
    Abstract [en]

    Resolution of branched DNA structures is pivotal for repair of stalled replication forks and meiotic recombination intermediates. The Yen1 nuclease cleaves both Holliday junctions and replication forks. We show that Yen1 interacts physically with Uls1, a suggested SUMO-targeted ubiquitin ligase that also contains a SWI/SNF-family ATPase-domain. Yen1 is SUMO-modified in its noncatalytic carboxyl terminus and DNA damage induces SUMOylation. SUMO-modification of Yen1 strengthens the interaction to Uls1, and mutations in SUMO interaction motifs in Uls1 weakens the interaction. However, Uls1 does not regulate the steady-state level of SUMO-modified Yen1 or chromatin-associated Yen1. In addition, SUMO-modification of Yen1 does not affect the catalytic activity in vitro. Consistent with a shared function for Uls1 and Yen1, mutations in both genes display similar phenotypes. Both uls1 and yen1 display negative genetic interactions with the alternative HJ-cleaving nuclease Mus81, manifested both in hypersensitivity to DNA damaging agents and in meiotic defects. Point mutations in ULS1 (uls1K975R and uls1C1330S, C1333S) predicted to inactivate the ATPase and ubiquitin ligase activities, respectively, are as defective as the null allele, indicating that both functions of Uls1 are essential. A micrococcal nuclease sequencing experiment showed that Uls1 had minimal effects on global nucleosome positioning/occupancy. Moreover, increased gene dosage of YEN1 partially alleviates the mus81 uls1 sensitivity to DNA damage. We suggest a preliminary model in which Uls1 acts in the same pathway as Yen1 to resolve branched DNA structures.

  • 6.
    Carter, Sidney D.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Vigasova, Dana
    Chen, Jiang
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Chovanec, Miroslav
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 29, p. 12037-12042Article in journal (Refereed)
    Abstract [en]

    Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair.

  • 7.
    Chen, Jiang
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Bauer, Stefanie
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    The helicase/SUMO-targeted ubiquitin ligase Uls1 interacts both physically and functionally with the Holliday junction resolvase Yen1Manuscript (preprint) (Other academic)
    Abstract [en]

    Yen1 is nuclease that can cleave the Holliday junction (HJ), an important DNA intermediate formed during homologous recombination. Here, we show that Yen1 interacts molecularly with Uls1, a SUMO targeted ubiquitin ligase that also belongs to the SWI/SNF-family of DNA-dependent ATPases. We demonstrate that Yen1 is SUMO modified in its carboxyl terminus and that this modification strengthens the interaction between Yen1 and Uls1. Absence of Uls1 increased the steady-state levels of Yen1, but only after extensive DNA damage, suggesting that Uls1 has a role in damage-induced degradation of Yen1. Consistent with a shared role for Uls1 and Yen1, mutations in the two enzymes display similar phenotypes. Both uls1 and yen1 have a negative genetic interaction with the alternative HJ-cleaving nuclease Mus81. This negative genetic interaction is manifested in supersensitivity to DNA damaging agents, but also in a meiotic defect. Neither mus81 uls1 nor mus81 yen1 double mutant diploids can complete meiosis. Moreover, both uls1 and yen1 exacerbates the chromosome mis-segregation phenotype of mus81. However, the mus81 uls1 yen1 triple mutant strain was slightly more sensitive to DNA damage compared to any double mutant combination, indicating that Uls1 and Yen1 also have independent roles in DNA repair. Point mutant alleles of Uls1 (uls1K975R and uls1C1330S/C1333S) that inactivates the ATPase and potential ubiquitin ligase activities are also supersensitive to DNA damage when combined with mus81, indicating that both activities of Uls1 are essential for function. We suggest that Yen1 and Uls1 are involved in an alternative pathway that is responsible for resolving complex DNA repair intermediates in the absence of Mus81.

  • 8.
    Chen, Jiang
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis2012In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 11, no 10, p. 833-843Article in journal (Refereed)
    Abstract [en]

    Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1Δ strain, which the yen1Δ allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms.

  • 9.
    Chen, Jiang
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Acetylation of the histone H3 N-terminus promotes DNA double-strand break repair in Kluyveromyces lactisManuscript (preprint) (Other academic)
    Abstract [en]

    Condensed chromatin hinders proteins from accessing the DNA, hence posing a block to processes like DNA repair. In this study, we investigate how histone modifications influence DNA double-strand break (DSB) repair. We show that blocking phosphorylation of serine 129 of histone H2A impairs DSB-repair, probably by reducing the efficiency of homologous recombination (HR). The lysine residues of histone H3 and H4 are subjected to reversible acetylation and methylation and we exchanged the lysines for either arginine (mimicking non-acetylated lysine) or glutamine (mimicking acetylated lysine). A histone H3 mutant with five N-terminal lysines exchanged for arginine showed reduced gene conversion and perturbed cell cycle progression. Leaving a single lysine residue intact was sufficient for protecting cells from DNA damage. In addition, exchanging the five lysines for glutamine did not result in these defects, indicating that one lysine residue in the histone H3 N-terminus must be acetylated for efficient DSB-repair. We find no evidence for that histone modification reduces the efficiency of nonhomologous end joining. Furthermore, the histone H3 K9, 14, 18, 23, 27R mutation is not defective in transcription of DSB repair genes indicating that the defects we observe in DSB-repair is unlikely to be due to indirect regulatory effects. These findings indicate that both histone H2A phosphorylation and histone H3 acetylation is important for the efficiency of the HR-pathway.

  • 10.
    Chiruvella, Kishore K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Rajaei, Naghmeh
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Jonna, Venkateswara Rao
    Hofer, Anders
    Åström, Stefan U.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 21671Article in journal (Refereed)
    Abstract [en]

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MAT alpha). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  • 11. Guerra, Lina
    et al.
    Guidi, Riccardo
    Slot, Ilse
    Callegari, Simone
    Sompallae, Ramakrishna
    Pickett, Carol L.
    Åström, Stefan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Eisele, Frederik
    Wolf, Dieter
    Sjögren, Camilla
    Masucci, Maria G.
    Frisan, Teresa
    Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival2011In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 124, no 16, p. 2735-2742Article in journal (Refereed)
    Abstract [en]

    The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.

  • 12.
    Rajaei, Naghmeh
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Chiruvella, Kishore K.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lin, Feng
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Åström, Stefan U.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 43, p. 15491-15496Article in journal (Refereed)
    Abstract [en]

    Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATa in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1-MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HML alpha) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MAT alpha locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed -1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATa genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase- derived endonuclease vital for sexual differentiation.

  • 13. Tsaponina, Olga
    et al.
    Barsoum, Emad
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Åström, Stefan U.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Developmental Biology.
    Chabes, Andrei
    Ixr1 Is Required for the Expression of the Ribonucleotide Reductase Rnr1 and Maintenance of dNTP Pools2011In: PLoS genetics, ISSN 1553-7390, Vol. 7, no 5, p. e1002061-Article in journal (Refereed)
    Abstract [en]

    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.

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