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  • 1. Bacal, Julien
    et al.
    Moriel-Carretero, María
    Pardo, Benjamin
    Barthe, Antoine
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lengronne, Armelle
    Pasero, Philippe
    Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage.2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, article id e99319Article in journal (Refereed)
    Abstract [en]

    The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.

  • 2. Barfeld, Stefan J
    et al.
    Fazli, Ladan
    Persson, Margareta
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Urbanucci, Alfonso
    Kaukoniemi, Kirsi M
    Rennie, Paul S
    Ceder, Yvonne
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Visakorpi, Tapio
    Mills, Ian G
    Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer2015In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 6, no 14, p. 12587-12602Article in journal (Refereed)
    Abstract [en]

    The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

  • 3.
    Buckland, Robert J
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Watt, Danielle L
    Chittoor, Balasubramanyam
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kunkel, Thomas A
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity2014In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, article id e1004846Article in journal (Refereed)
    Abstract [en]

    The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

  • 4.
    Chabes, Andrei
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liu, Aimin
    Gräslund, Astrid
    Wijmenga, Sybren S
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit2000In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 97, no 6, p. 2474-2479Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Eukaryotes have an alpha(2)beta(2) form of RNR consisting of two homodimeric subunits, proteins R1 (alpha(2)) and R2 (beta(2)). The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors. The R2 protein is a radical storage device containing an iron center-generated tyrosyl free radical. Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding. Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p (beta') is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p/Rnr4p complex. This complex sediments at 5.6 S as a betabeta' heterodimer in a sucrose gradient. In the presence of Rnr1p, both polypeptides of the Rnr2p/Rnr4p heterodimer cosediment at 9.7 S as expected for an alpha(2)betabeta' heterotetramer, where Rnr4p plays an important role in the interaction between the alpha(2) and the betabeta ' subunits. The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5'-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity. This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy. Taken together, our results show that the Rnr2p/Rnr4p heterodimer is the active form of the yeast RNR small subunit.

  • 5.
    Chabes, Andrei
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Georgieva, Bilyana
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhao, Xiaolan
    Rothstein, Rodney
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase.2003In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 112, no 3, p. 391-401Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Chabes, Andrei
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Stillman, Bruce
    Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae.2007In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 104, no 4, p. 1183-8Article in journal (Refereed)
  • 7.
    Chabes, Andrei
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA building blocks at the foundation of better survival.2003In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 2, no 3, p. 171-3Article in journal (Refereed)
  • 8. Coquel, Flavie
    et al.
    Silva, Maria-Joao
    Técher, Hervé
    Zadorozhny, Karina
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nieminuszczy, Jadwiga
    Mettling, Clément
    Dardillac, Elodie
    Barthe, Antoine
    Schmitz, Anne-Lyne
    Promonet, Alexy
    Cribier, Alexandra
    Sarrazin, Amélie
    Niedzwiedz, Wojciech
    Lopez, Bernard
    Costanzo, Vincenzo
    Krejci, Lumir
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Benkirane, Monsef
    Lin, Yea-Lih
    Pasero, Philippe
    SAMHD1 acts at stalled replication forks to prevent interferon induction2018In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 557, no 7703, p. 57-61Article in journal (Refereed)
    Abstract [en]

    SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.

  • 9. Davidson, Marta B.
    et al.
    Katou, Yuki
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sing, Tina L.
    Xia, Tian
    Ou, Jiongwen
    Vaisica, Jessica A.
    Thevakumaran, Neroshan
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Myers, Chad L.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Shirahige, Katsuhiko
    Brown, Grant W.
    Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 4, p. 895-907Article in journal (Refereed)
    Abstract [en]

    The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis. The EMBO Journal (2012) 31, 895-907. doi: 10.1038/emboj.2011.485; Published online 10 January 2012

  • 10. Deem, A
    et al.
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blackgrove, T
    Vayl, A
    Coffey, B
    Mathur, R
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Malkova, A
    Department of Biology, School of Science, IUPUI, Indianapolis, Indiana, United States of America.
    Break-induced replication is highly inaccurate2011In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885Article in journal (Refereed)
    Abstract [en]

    DNA must be synthesized for purposes of genome duplication and DNA repair. While the former is a highly accurate process, short-patch synthesis associated with repair of DNA damage is often error-prone. Break-induced replication (BIR) is a unique cellular process that mimics normal DNA replication in its processivity, rate, and capacity to duplicate hundreds of kilobases, but is initiated at double-strand breaks (DSBs) rather than at replication origins. Here we employed a series of frameshift reporters to measure mutagenesis associated with BIR in Saccharomyces cerevisiae. We demonstrate that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2,800-fold higher than during normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. We established that polymerase proofreading and mismatch repair correct BIR errors. Also, dNTP levels were elevated during BIR, and this contributed to BIR-related mutagenesis. We propose that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR, with Pol δ generating many of the mutagenic errors. We further postulate that activation of BIR in eukaryotic cells may significantly contribute to accumulation of mutations that fuel cancer and evolution.

  • 11.
    Domkin, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Phosphines are ribonucleotide reductase reductants that act via C-terminal cysteines similar to thioredoxins and glutaredoxins2014In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 4, p. 5539-Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the formation of 2'-deoxyribonucleotides. Each polypeptide of the large subunit of eukaryotic RNRs contains two redox-active cysteine pairs, one in the active site and the other at the C-terminus. In each catalytic cycle, the active-site disulfide is reduced by the C-terminal cysteine pair, which in turn is reduced by thioredoxins or glutaredoxins. Dithiols such as DTT are used in RNR studies instead of the thioredoxin or glutaredoxin systems. DTT can directly reduce the disulfide in the active site and does not require the C-terminal cysteines for RNR activity. Here we demonstrate that the phosphines tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THP) are efficient non-thiol RNR reductants, but in contrast to the dithiols DTT, bis(2-mercaptoethyl)sulfone (BMS), and (S)-(1,4-dithiobutyl)-2-amine (DTBA) they act specifically via the C-terminal disulfide in a manner similar to thioredoxin and glutaredoxin. The simultaneous use of phosphines and dithiols results in ~3-fold higher activity compared to what is achieved when either type of reductant is used alone. This surprising effect can be explained by the concerted action of dithiols on the active-site cysteines and phosphines on the C-terminal cysteines. As non-thiol and non-protein reductants, phosphines can be used to differentiate between the redox-active cysteine pairs in RNRs.

  • 12.
    Domkin, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 21, p. 18574-8Article in journal (Refereed)
    Abstract [en]

    The ribonucleotide reductase system in Saccharomyces cerevisiae includes four genes (RNR1 and RNR3 encoding the large subunit and RNR2 and RNR4 encoding the small subunit). RNR3 expression, nearly undetectable during normal growth, is strongly induced by DNA damage. Yet an rnr3 null mutant has no obvious phenotype even under DNA damaging conditions, and the contribution of RNR3 to ribonucleotide reduction is not clear. To investigate the role of RNR3 we expressed and characterized the Rnr3 protein. The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. However, a strong synergism between Rnr3 and Rnr1 was observed, most clearly demonstrated in experiments with the catalytically inactive Rnr1-C428A mutant, which increased the endogenous activity of Rnr3 by at least 10-fold. In vivo, the levels of Rnr3 after DNA damage never reached more than one-tenth of the Rnr1 levels. We propose that heterodimerization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the ribonucleotide reductase holoenzyme, which may be important when Rnr1 is limiting for dNTP production. In complex with inactive Rnr1-C428A, the activity of Rnr3 is controlled by effector binding to Rnr1-C428A. This result indicates cross-talk between the Rnr1 and Rnr3 polypeptides of the large subunit.

  • 13. Earp, Caroline
    et al.
    Rowbotham, Samuel
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cha, Rita S
    Acute sensitivity of DNA replication to reduction in dNTP pools following Mec1ATR inactivation2015In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, no Suppl. 1, p. S56-S56Article in journal (Other academic)
    Abstract [en]

    Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by a fatal mitotic catastrophe. The mec1 S phase arrest was proposed to stem from a defect in the Mec1-Rad53-Dun1 dependent removal of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), at the onset of S phase: According to this view, Sml1 removal and the ensuing RNR activation would promote the dNTP production necessary for genome duplication. In support for this view, dNTP levels in hypomorphic mec1 or rad53 mutants and a dun1∆ strain were shown to be reduced by as much as 46% compared to a MEC1 control strain. Notably however, nearly all analyses on a lethal mec1 allele (e.g. mec1∆ or mec1-kd [kinase dead]) have been performed in a strain background that was either deleted for SML1 or over-expressing RNR1, a requirement for maintaining viability of a mutant lacking Mec1's essential function. As a result, while it is clear that absence of Mec1 causes dNTP pool to decrease, the true extent of the reduction and whether it would be sufficient to account for the replication arrest remain elusive. Here, we addressed these questions utilizing a temperature sensitive mutant, mec1-4, which maintains its viability at permissive temperature in an otherwise wild-type background, circumventing the need to exogenously manipulate Sml1 and/or RNR activity. Results show that Mec1 inactivation leads to an S phase arrest and a ~17% reduction in dNTP pool; expression of a novel suppressor, GIS2 (glucose inhibition of gluconeogenic growth suppressor 2), rescues the arrest and partially restores the dNTP pool to ~ 93% of a control. Unexpectedly modest effects of mec1 and GIS2 on dNTP levels demonstrate that the arrest does result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to its availability

  • 14. Earp, Caroline
    et al.
    Rowbotham, Samuel
    Merényi, Gábor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Cha, Rita S
    S phase block following MEC1ATR inactivation occurs without severe dNTP depletion2015In: Biology open, ISSN 2046-6390, Vol. 4, no 12, p. 1739-1743Article in journal (Refereed)
    Abstract [en]

    Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

  • 15. Fasullo, Michael
    et al.
    Tsaponina, Olga
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sun, Mingzeng
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elevated dNTP levels suppress hyper-recombination in Saccharomyces cerevisiae S-phase checkpoint mutants2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 4, p. 1195-1203Article in journal (Refereed)
    Abstract [en]

    MEC1, the essential yeast homolog of the human ATR/ATM genes, controls the S-phase checkpoint and prevents replication fork collapse at slow zones of DNA replication. The viability of hypomorphic mec1-21 is reduced in the rad52 mutant, defective in homologous recombination, suggesting that replication generates recombinogenic lesions. We previously observed a 6-, 10- and 30-fold higher rate of spontaneous sister chromatid exchange (SCE), heteroallelic recombination and translocations, respectively, in mec1-21 mutants compared to wild-type. Here we report that the hyper-recombination phenotype correlates with lower deoxyribonucleoside triphosphate (dNTP) levels, compared to wild-type. By introducing a dun1 mutation, thus eliminating inducible expression of ribonucleotide reductase in mec1-21, rates of spontaneous SCE increased 15-fold above wild-type. All the hyper-recombination phenotypes were reduced by SML1 deletions, which increase dNTP levels. Measurements of dNTP pools indicated that, compared to wild-type, there was a significant decrease in dNTP levels in mec1-21, dun1 and mec1-21 dun1, while the dNTP levels of mec1-21 sml1, mec1-21 dun1 sml1 and sml1 mutants were approximately 2-fold higher. Interestingly, higher dNTP levels in mec1-21 dun1 sml1 correlate with approximately 2-fold higher rate of spontaneous mutagenesis, compared to mec1-21 dun1. We suggest that higher dNTP levels in specific checkpoint mutants suppress the formation of recombinogenic lesions.

  • 16. Gan, Haiyun
    et al.
    Yu, Chuanhe
    Devbhandari, Sujan
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Han, Junhong
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Remus, Dirk
    Zhang, Zhiguo
    Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress2017In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, no 2, p. 446-455Article in journal (Refereed)
    Abstract [en]

    The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.

  • 17. Garzón, J.
    et al.
    Rodríguez, R.
    Kong, Ziqing
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rodríguez-Acebes, S.
    Méndez, J.
    Moreno, S.
    García-Higuera, I.
    Shortage of dNTPs underlies altered replication dynamics and DNA breakage in the absence of the APC/C cofactor Cdh12017In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 36, no 42, p. 5808-5818Article in journal (Refereed)
    Abstract [en]

    The APC/C-Cdh1 ubiquitin-ligase complex targets cell cycle regulators for proteosomal degradation and helps prevent tumor development and accumulation of chromosomal aberrations. Replication stress has been proposed to be the main driver of genomic instability in the absence of Cdh1, but the real contribution of APC/C-Cdh1 to efficient replication, especially in normal cells, remains unclear. Here we show that, in primary MEFs, acute depletion or permanent ablation of Cdh1 slowed down replication fork movement and increased origin activity. Partial inhibition of origin firing does not accelerate replication forks, suggesting that fork progression is intrinsically limited in the absence of Cdh1. Moreover, exogenous supply of nucleotide precursors, or ectopic overexpression of RRM2, the regulatory subunit of Ribonucleotide Reductase, restore replication efficiency, indicating that dNTP availability could be impaired upon Cdh1 loss. Indeed, we found reduced dNTP levels in Cdh1-deficient MEFs. Importantly, DNA breakage is also significantly alleviated by increasing intracellular dNTP pools, strongly suggesting that genomic instability is the result of aberrant replication. These observations highlight the relevance of APC/C-Cdh1 activity during G1 to ensure an adequate supply of dNTPs to the replisome, prevent replication stress and the resulting chromosomal breaks and, ultimately, suppress tumorigenesis.

  • 18. Gupta, Amitabha
    et al.
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Reichenbach, Patrick
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lingner, Joachim
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rothstein, Rodney
    Chang, Michael
    Telomere Length Homeostasis Responds to Changes in Intracellular dNTP Pools2013In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 193, no 4, p. 1095-1105Article in journal (Refereed)
    Abstract [en]

    Telomeres, the ends of linear eukaryotic chromosomes, shorten due to incomplete DNA replication and nucleolytic degradation. Cells counteract this shortening by employing a specialized reverse transcriptase called telomerase, which uses deoxyribonucleoside triphosphates (dNTPs) to extend telomeres. Intracellular dNTP levels are tightly regulated and perturbation of these levels is known to affect DNA synthesis. We examined whether altering the levels of the dNTP pools or changing the relative ratios of the four dNTPs in Saccharomyces cerevisiae would affect the length of the telomeres. Lowering dNTP levels leads to a modest shortening of telomeres, while increasing dNTP pools has no significant effect on telomere length. Strikingly, altering the ratio of the four dNTPs dramatically affects telomere length homeostasis, both positively and negatively. Specifically, we find that intracellular dGTP levels positively correlate with both telomere length and telomerase nucleotide addition processivity in vivo. Our findings are consistent with in vitro data showing dGTP-dependent stimulation of telomerase activity in multiple organisms, and suggest that telomerase activity is modulated in vivo by dGTP levels.

  • 19. Hoch, Nicolas C
    et al.
    Chen, Eric S-W
    Buckland, Robert
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wang, Shun-Chung
    Fazio, Alessandro
    Hammet, Andrew
    Pellicioli, Achille
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Tsai, Ming-Daw
    Heierhorst, Jörg
    Molecular basis of the essential s phase function of the rad53 checkpoint kinase2013In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 33, no 16, p. 3202-3213Article in journal (Refereed)
    Abstract [en]

    The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.

  • 20.
    Hofer, Anders
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Steverding, D
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Brun, R
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness.2001In: Proceedings of the National Academy of Sciences of the U S A, ISSN 0027-8424, Vol. 98, no 11, p. 6412-6Article in journal (Refereed)
  • 21.
    Jia, Shaodong
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Buckland, Robert
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection2015In: DNA Replikation: Methods and Protocols / [ed] Sonya Vengrova; Jacob Dalgaard, Warwick Medical School, University of Warwick, New York: Springer-Verlag New York, 2015, Vol. 1300, p. 113-121Chapter in book (Refereed)
    Abstract [en]

    DNA polymerase assays are commonly used for the detection of deoxyribonucleoside triphosphates (dNTPs) in biological samples. For better specificity and accuracy, high-performance liquid chromatography (HPLC) methods have been developed for the analysis of the four dNTPs in complex samples. Here we describe a simple method using isocratic strong anion-exchange (SAX) chromatographic separation coupled with ultraviolet detection (UV) for the analysis of the four dNTPs in budding yeast Saccharomyces cerevisiae. This method can be applied to other species of yeast or bacteria.

  • 22.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Speck, Christian
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    A top-down view on DNA replication and recombination from 9,000 feet above sea level2011In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 12, no 4, article id 304Article in journal (Refereed)
    Abstract [en]

    A report of the Keystone Symposium 'DNA Replication and Recombination' held in Keystone, USA, 27 February to 4 March 2011.

  • 23. Kenigsberg, Ephraim
    et al.
    Yehuda, Yishai
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tanay, Amos
    Simon, Itamar
    The mutation spectrum in genomic late replication domains shapes mammalian GC content2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 9, p. 4222-4232Article in journal (Refereed)
    Abstract [en]

    Genome sequence compositions and epigenetic organizations are correlated extensively across multiple length scales. Replication dynamics, in particular, is highly correlated with GC content. We combine genome-wide time of replication (ToR) data, topological domains maps and detailed functional epigenetic annotations to study the correlations between replication timing and GC content at multiple scales. We find that the decrease in genomic GC content at large scale late replicating regions can be explained by mutation bias favoring A/T nucleotide, without selection or biased gene conversion. Quantification of the free dNTP pool during the cell cycle is consistent with a mechanism involving replication-coupled mutation spectrum that favors AT nucleotides at late S-phase. We suggest that mammalian GC content composition is shaped by independent forces, globally modulating mutation bias and locally selecting on functional element. Deconvoluting these forces and analyzing them on their native scales is important for proper characterization of complex genomic correlations.

  • 24. Kochenova, Olga V
    et al.
    Bezalel-Buch, Rachel
    Tran, Phong
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Makarova, Alena V
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Burgers, Peter M J
    Shcherbakova, Polina V
    Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 3, p. 1200-1218Article in journal (Refereed)
    Abstract [en]

    In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at 'normal S-phase' and 'damage-response' dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three 'X-dCTP' mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from 'S-phase' to 'damage-response' dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.

  • 25.
    Kong, Ziqing
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jia, Shaodong
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Anna Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Appelblad, Patrik
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience. Merck Chemicals and Life Science AB, Solna, Sweden.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Moritz, Thomas
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 11, article id e66Article in journal (Refereed)
    Abstract [en]

    Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.

  • 26.
    Kumar, Dinesh
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Abdulovic, Amy L
    Viberg, Jörgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kunkel, Thomas A
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 4, p. 1360-1371Article in journal (Refereed)
    Abstract [en]

    The mechanisms by which imbalanced dNTPs induce mutations have been well characterized within a test tube, but not in vivo. We have examined mechanisms by which dNTP imbalances induce genome instability in strains of Saccharomyces cerevisiae with different amino acid substitutions in Rnr1, the large subunit of ribonucleotide reductase. These strains have different dNTP imbalances that correlate with elevated CAN1 mutation rates, with both substitution and insertion-deletion rates increasing by 10- to 300-fold. The locations of the mutations in a strain with elevated dTTP and dCTP are completely different from those in a strain with elevated dATP and dGTP. Thus, imbalanced dNTPs reduce genome stability in a manner that is highly dependent on the nature and degree of the imbalance. Mutagenesis is enhanced despite the availability of proofreading and mismatch repair. The mutations can be explained by imbalanced dNTP-induced increases in misinsertion, strand misalignment and mismatch extension at the expense of proofreading. This implies that the relative dNTP concentrations measured in extracts are truly available to a replication fork in vivo. An interesting mutational strand bias is observed in one rnr1 strain, suggesting that the S-phase checkpoint selectively prevents replication errors during leading strand replication.

  • 27.
    Kumar, Dinesh
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Viberg, Jörgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 12, p. 3975-3983Article in journal (Refereed)
    Abstract [en]

    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.

  • 28. Lanz, Michael Charles
    et al.
    Oberly, Susannah
    Sanford, Ethan James
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Smolka, Marcus Bustamante
    Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling2018In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 32, no 11-12, p. 822-835Article in journal (Refereed)
    Abstract [en]

    The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of "free" Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, "free" MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.

  • 29. Li, Shuqi
    et al.
    Xu, Zhiyun
    Xu, Jiawei
    Zuo, Linyu
    Yu, Chuanhe
    Zheng, Pu
    Gan, Haiyun
    Wang, Xuezheng
    Li, Longtu
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Li, Di
    Wang, Sheng
    Zheng, Sihao
    Li, Jinbao
    Chen, Xuefeng
    Sun, Yujie
    Xu, Dongyi
    Han, Junhong
    Chan, Kuiming
    Qi, Zhi
    Feng, Jianxun
    Li, Qing
    Rtt105 functions as a chaperone for replication protein A to preserve genome stability2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 17, article id e99154Article in journal (Refereed)
    Abstract [en]

    Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.

  • 30. Liew, Li Phing
    et al.
    Lim, Zun Yi
    Cohen, Matan
    Kong, Ziqing
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bell, Stephen D
    Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase2016In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 17, no 6, p. 1657-1670Article in journal (Refereed)
    Abstract [en]

    In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment.

  • 31. Lin, Chia-Yeh
    et al.
    Wu, Meng-Ying
    Gay, Sophie
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lai, Mong Sing
    Hsiao, Wei-Chun
    Hung, Shih-Hsun
    Tseng, Hsin-Yi
    Wright, Duncan Edward
    Wang, Chen-Yi
    Hsu, Guoo-Shyng W
    Devys, Didier
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kao, Cheng-Fu
    H2B Mono-ubiquitylation Facilitates Fork Stalling and Recovery during Replication Stress by Coordinating Rad53 Activation and Chromatin Assembly2014In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 10, p. e1004667-Article in journal (Refereed)
    Abstract [en]

    The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

  • 32.
    Maicher, André
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gazy, Inbal
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Grinberg, Gilad
    Shemesh, Keren
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kupiec, Martin
    Rnr1, but not Rnr3, facilitates the sustained telomerase-dependent elongation of telomeres2017In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 13, no 10, article id e1007082Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) provides the precursors for the generation of dNTPs, which are required for DNA synthesis and repair. Here, we investigated the function of the major RNR subunits Rnr1 and Rnr3 in telomere elongation in budding yeast. We show that Rnr1 is essential for the sustained elongation of short telomeres by telomerase. In the absence of Rnr1, cells harbor very short, but functional, telomeres, which cannot become elongated by increased telomerase activity or by tethering of telomerase to telomeres. Furthermore, we demonstrate that Rnr1 function is critical to prevent an early onset of replicative senescence and premature survivor formation in telomerase-negative cells but dispensable for telomere elongation by Homology-Directed-Repair. Our results suggest that telomerase has a "basal activity" mode that is sufficient to compensate for the "end-replication-problem" and does not require the presence of Rnr1 and a different "sustained activity" mode necessary for the elongation of short telomeres, which requires an upregulation of dNTP levels and dGTP ratios specifically through Rnr1 function. By analyzing telomere length and dNTP levels in different mutants showing changes in RNR complex composition and activity we provide evidence that the Mec1ATR checkpoint protein promotes telomere elongation by increasing both dNTP levels and dGTP ratios through Rnr1 upregulation in a mechanism that cannot be replaced by its homolog Rnr3.

  • 33.
    Mertz, Tony M
    et al.
    Omaha, Nebraska, USA.
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Shcherbakova, Polina V
    Omaha, Nebraska, USA.
    Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 19, p. E2467-E2476Article in journal (Refereed)
    Abstract [en]

    Defects in DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.

  • 34. Nick McElhinny, Stephanie A
    et al.
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Clark, Alan B
    Watt, Danielle L
    Watts, Brian E
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kunkel, Thomas A
    Genome instability due to ribonucleotide incorporation into DNA2010In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 6, no 10, p. 774-81Article in journal (Refereed)
    Abstract [en]

    Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase ϵ (Pol ϵ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol ϵ compared to wild-type Pol ϵ. This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S phase, had elevated dNTP pools and generated 2-5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

  • 35. Nick McElhinny, Stephanie A
    et al.
    Watts, Brian E
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Watt, Danielle L
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M J
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kunkel, Thomas A
    Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 11, p. 4949-4954Article in journal (Refereed)
    Abstract [en]

    Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36- to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase epsilon, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol delta and Pol alpha, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols alpha, delta, and epsilon, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase epsilon has difficulty bypassing a single rNMP present within a DNA template.

  • 36. Nystrom, K.
    et al.
    Pettersson, G.
    Wanrooij, Paulina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brunet, S.
    Said, J.
    Ortolani, G.
    Waldenstrom, J.
    Adamek, L.
    Tang, K. -W
    Norberg, P.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hellstrand, K.
    Norder, H.
    Lagging, M.
    Inosine triphosphate pyrophosphatase enhances the effect of ribavirin on hepatitis C virus cell culture infection2017In: Journal of Hepatology, ISSN 0168-8278, E-ISSN 1600-0641, Vol. 66, no 1, p. S321-S321Article in journal (Refereed)
    Abstract [en]

    Genetic variants of the inosine triphosphate pyrophosphatase gene (ITPA), resulting in decreased enzymatic activity of the corresponding enzyme, ITPase, are known to correlate with a decreased risk of ribavirin-induced anemia, but are also associated with an increased SVR in patients treated with peginterferon-alpha and ribavirin. As both ITPase and ribavirin are involved in the nucleotide salvage pathway and reduced risk of relapse after treatment of hepatitis C, we have investigated the effect of ITPase activity and ribavirin treatment of HCVcc infection of hepatocytes

  • 37. Peng, Xiao P.
    et al.
    Lim, Shelly
    Li, Shibai
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhao, Xiaolan
    Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites2018In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 1, article id e1007129Article in journal (Refereed)
    Abstract [en]

    Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA) array. Each rDNA repeat contains a programmed replication fork barrier (RFB) established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1 Delta and fob1 Delta similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.

  • 38. Poli, Jerome
    et al.
    Tsaponina, Olga
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Crabbe, Laure
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pantesco, Veronique
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lengronne, Armelle
    Pasero, Philippe
    dNTP pools determine fork progression and origin usage under replication stress2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 4, p. 883-894Article in journal (Refereed)
    Abstract [en]

    Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

  • 39.
    Rentoft, Matilda
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindell, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tran, Phong
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Anna Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Buckland, Robert
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Watt, Danielle L.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Melin, Beatrice
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 17, p. 4723-4728Article in journal (Refereed)
    Abstract [en]

    Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that severalSAMHD1mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of oneSAMHD1allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associatedSAMHD1mutations increase mutation rates in cancer cells.

  • 40.
    Rossmann, Marlies P
    et al.
    Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
    Luo, Weijun
    Tsaponina, Olga
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Stillman, Bruce
    Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
    A common telomeric gene silencing assay is affected by nucleotide metabolism2011In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 42, no 1, p. 127-136Article in journal (Refereed)
    Abstract [en]

    Telomere-associated position-effect variegation (TPEV) in budding yeast has been used as a model for understanding epigenetic inheritance and gene silencing. A widely used assay to identify mutants with improper TPEV employs the URA3 gene at the telomere of chromosome VII-L that can be counterselected with 5-fluoroorotic acid (5-FOA). 5-FOA resistance has been inferred to represent lack of transcription of URA3 and therefore to represent heterochromatin-induced gene silencing. For two genes implicated in telomere silencing, POL30 and DOT1, we show that the URA3 telomere reporter assay does not reflect their role in heterochromatin formation. Rather, an imbalance in ribonucleotide reductase (RNR), which is induced by 5-FOA, and the specific promoter of URA3 fused to ADH4 at telomere VII-L are jointly responsible for the variegated phenotype. We conclude that metabolic changes caused by the drug employed and certain mutants being studied are incompatible with the use of certain prototrophic markers for TPEV.

  • 41. Rubinstein, Linda
    et al.
    Ungar, Lior
    Harari, Yaniv
    Babin, Vera
    Ben-Aroya, Shay
    Merenyi, Gabor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kupiec, Martin
    Telomere length kinetics assay (TELKA) sorts the telomere length maintenance (tlm) mutants into functional groups2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 10, p. 6314-6325Article in journal (Refereed)
    Abstract [en]

    Genome-wide systematic screens in yeast have uncovered a large gene network (the telomere length maintenance network or TLM), encompassing more than 400 genes, which acts coordinatively to maintain telomere length. Identifying the genes was an important first stage; the next challenge is to decipher their mechanism of action and to organize then into functional groups or pathways. Here we present a new telomere-length measuring program, TelQuant, and a novel assay, telomere length kinetics assay, and use them to organize tlm mutants into functional classes. Our results show that a mutant defective for the relatively unknown MET7 gene has the same telomeric kinetics as mutants defective for the ribonucleotide reductase subunit Rnr1, in charge of the limiting step in dNTP synthesis, or for the Ku heterodimer, a well-established telomere complex. We confirm the epistatic relationship between the mutants and show that physical interactions exist between Rnr1 and Met7. We also show that Met7 and the Ku heterodimer affect dNTP formation, and play a role in non-homologous end joining. Thus, our telomere kinetics assay uncovers new functional groups, as well as complex genetic interactions between tlm mutants.

  • 42.
    Sabouri, Nasim
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Viberg, Jörgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 17, p. 5660-5667Article in journal (Refereed)
    Abstract [en]

    The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases approximately 6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase epsilon does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19-27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions.

  • 43. Saitoh, Shigeaki
    et al.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    McDonald, W Hayes
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yates, John R
    Russell, Paul
    Cid13 is a cytoplasmic poly(A) polymerase that regulates ribonucleotide reductase mRNA.2002In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 109, no 5, p. 563-73Article in journal (Refereed)
    Abstract [en]

    Fission yeast Cid13 and budding yeast Trf4/5 are members of a newly identified nucleotidyltransferase family conserved from yeast to man. Trf4/5 are thought to be essential DNA polymerases. We report that Cid13 is a poly(A) polymerase. Unlike conventional poly(A) polymerases, which act in the nucleus and indiscriminately polyadenylate all mRNA, Cid13 is a cytoplasmic enzyme that specifically targets suc22 mRNA that encodes a subunit of ribonucleotide reductase (RNR). cid13 mutants have reduced dNTP pools and are sensitive to hydroxyurea, an RNR inhibitor. We propose that Cid13 defines a cytoplasmic form of poly(A) polymerase important for DNA replication and genome maintenance.

  • 44. Schmidt, Tobias T
    et al.
    Reyes, Gloria
    Gries, Kerstin
    Ceylan, Cemile Ümran
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Meurer, Matthias
    Knop, Michael
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hombauer, Hans
    Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 22, p. E4442-E4451Article in journal (Refereed)
    Abstract [en]

    Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in Saccharomyces cerevisiae using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (GLN3, URA7, and SHM2) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.

  • 45. Schmidt, Tobias T.
    et al.
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Reyes, Gloria X.
    Gries, Kerstin
    Gross, Maike
    Zhao, Boyu
    Yuan, Jui-Hung
    Wade, Rebecca
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hombauer, Hans
    A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
    Abstract [en]

    The balance and the overall concentration of intracellular deoxyribonucleoside triphosphates (dNTPs) are important determinants of faithful DNA replication. Despite the established fact that changes in dNTP pools negatively influence DNA replication fidelity, it is not clear why certain dNTP pool alterations are more mutagenic than others. As intracellular dNTP pools are mainly controlled by ribonucleotide reductase (RNR), and given the limited number of eukaryotic RNR mutations characterized so far, we screened for RNR1 mutations causing mutator phenotypes in Saccharomyces cerevisiae. We identified 24 rnr1 mutant alleles resulting in diverse mutator phenotypes linked in most cases to imbalanced dNTPs. Among the identified rnr1 alleles the strongest mutators presented a dNTP imbalance in which three out of the four dNTPs were elevated (dCTP, dTTP and dGTP), particularly if dGTP levels were highly increased. These rnr1 alleles caused growth defects/lethality in DNA replication fidelity-compromised backgrounds, and caused strong mutator phenotypes even in the presence of functional DNA polymerases and mismatch repair. In summary, this study pinpoints key residues that contribute to allosteric regulation of RNR’s overall activity or substrate specificity. We propose a model that distinguishes between different dNTP pool alterations and provides a mechanistic explanation why certain dNTP imbalances are particularly detrimental.

  • 46. Stone, Jana E
    et al.
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Binz, Sara K
    Inase, Aki
    Iwai, Shigenori
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Burgers, Peter M
    Kunkel, Thomas A
    Lesion bypass by S. cerevisiae Pol ζ alone2011In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 10, no 8, p. 826-834Article in journal (Refereed)
    Abstract [en]

    DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27 and 73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS.

  • 47.
    Sánchez, Arancha
    et al.
    Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA .
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rozenzhak, Sophie
    Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA .
    Roguev, Assen
    Univ Calif San Francisco, Dept Cellular & Mol Pharmacol, San Francisco, CA 94143 USA .
    Krogan, Nevan J
    Univ Calif San Francisco, Dept Cellular & Mol Pharmacol, San Francisco, CA 94143 USA .
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Russell, Paul
    Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA .
    Replication fork collapse and genome instability in dCMP deaminase mutant2012In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 32, no 21, p. 4445-4454Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δcells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2) and Chk1, and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.

  • 48.
    Tsaponina, Olga
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Barsoum, Emad
    Åström, Stefan U
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ixr1 is required for the expression of the ribonucleotide reductase Rnr1 and maintenance of dNTP pools2011In: PLoS genetics, ISSN 1553-7404, 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.

  • 49.
    Tsaponina, Olga
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Pre-activation of the genome integrity checkpoint increases DNA damage tolerance2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 22, p. 10371-10378Article in journal (Refereed)
    Abstract [en]

    The genome integrity checkpoint is a conserved signaling pathway that is regulated in yeast by the Mec1 (homologous to human ATR) and Rad53 (homologous to human Chk1) kinases. The pathway coordinates a multifaceted response that allows cells to cope with DNA damage and DNA replication stress. The full activation of the checkpoint blocks origin firing, stabilizes replication forks, activates DNA repair proteins and may lead to senescence or apoptosisin higher eukaryotes. We have recently demonstrated that endogenous replication stress can activate the genome integrity checkpoint in budding yeast at a low level that does not go so far as to interfere with cell cycle progression, but it does activate DNA damage-inducible proteins. Here we demonstrate that the low level pre-activation of the checkpoint, either by endogenous replication stress or by the nucleotide-depleting drug hydroxyurea, can increase damage tolerance to multiple DNA-damaging agents. These results may provide new strategies for using the checkpoint to protect normal cells from genotoxic stress.

  • 50. van Mourik, Paula M
    et al.
    de Jong, Jannie
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kavšek, Alan
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Chang, Michael
    Upregulation of dNTP Levels After Telomerase Inactivation Influences Telomerase-Independent Telomere Maintenance Pathway Choice in Saccharomyces cerevisiae2018In: G3: Genes, Genomes, Genetics, ISSN 2160-1836, E-ISSN 2160-1836, Vol. 8, no 8, p. 2551-2558Article in journal (Refereed)
    Abstract [en]

    In 10–15% of cancers, telomere length is maintained by a telomerase-independent, recombination-mediated pathway called alternative lengthening of telomeres (ALT). ALT mechanisms were first seen, and have been best studied, in telomerase-null Saccharomyces cerevisiae cells called “survivors”. There are two main types of survivors. Type I survivors amplify Y′ subtelomeric elements while type II survivors, similar to the majority of human ALT cells, amplify the terminal telomeric repeats. Both types of survivors require Rad52, a key homologous recombination protein, and Pol32, a non-essential subunit of DNA polymerase δ. A number of additional proteins have been reported to be important for either type I or type II survivor formation, but it is still unclear how these two pathways maintain telomeres. In this study, we performed a genome-wide screen to identify novel genes that are important for the formation of type II ALT-like survivors. We identified 23 genes that disrupt type II survivor formation when deleted. 17 of these genes had not been previously reported to do so. Several of these genes (DUN1CCR4, and MOT2) are known to be involved in the regulation of dNTP levels. We find that dNTP levels are elevated early after telomerase inactivation and that this increase favors the formation of type II survivors.

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