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  • 1. Bacal, Julien
    et al.
    Moriel-Carretero, María
    Pardo, Benjamin
    Barthe, Antoine
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lengronne, Armelle
    Pasero, Philippe
    Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage2018Inngår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, nr 21, artikkel-id e99319Artikkel i tidsskrift (Fagfellevurdert)
    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. Bru, Samuel
    et al.
    Marc Martinez-Lainez, Joan
    Hernandez-Ortega, Sara
    Quandt, Eva
    Torres-Torronteras, Javier
    Marti, Ramon
    Canadell, David
    Arino, Joaquin
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Jimenez, Javier
    Clotet, Josep
    Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae2016Inngår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 101, nr 3, s. 367-380Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4 Delta) or hydrolyse polyP (ppn1 Delta, ppx1 Delta) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.

  • 3. Cohen, Rotem
    et al.
    Milo, Shira
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Savidor, Alon
    Covo, Shay
    Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress2019Inngår i: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 83, artikkel-id 102674Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed.

  • 4. Coquel, Flavie
    et al.
    Silva, Maria-Joao
    Técher, Hervé
    Zadorozhny, Karina
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (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 universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Benkirane, Monsef
    Lin, Yea-Lih
    Pasero, Philippe
    SAMHD1 acts at stalled replication forks to prevent interferon induction2018Inngår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 557, nr 7703, s. 57-61Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 5. Gan, Haiyun
    et al.
    Yu, Chuanhe
    Devbhandari, Sujan
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Han, Junhong
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Remus, Dirk
    Zhang, Zhiguo
    Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress2017Inngår i: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, nr 2, s. 446-455Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 6. Garbacz, Marta A.
    et al.
    Cox, Phillip B.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lujan, Scott A.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kunkel, Thomas A.
    The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity2019Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 8, s. 3986-3995Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.

  • 7. Gupta, Amitabha
    et al.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Reichenbach, Patrick
    Marjavaara, Lisette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Anna Karin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lingner, Joachim
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rothstein, Rodney
    Chang, Michael
    Telomere Length Homeostasis Responds to Changes in Intracellular dNTP Pools2013Inngår i: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 193, nr 4, s. 1095-1105Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 8.
    Jia, Shaodong
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Marjavaara, Lisette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Buckland, Robert
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection2015Inngår i: 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, s. 113-121Kapittel i bok, del av antologi (Fagfellevurdert)
    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.

  • 9. Lanz, Michael Charles
    et al.
    Oberly, Susannah
    Sanford, Ethan James
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Smolka, Marcus Bustamante
    Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling2018Inngår i: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 32, nr 11-12, s. 822-835Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 10. Li, Shuqi
    et al.
    Xu, Zhiyun
    Xu, Jiawei
    Zuo, Linyu
    Yu, Chuanhe
    Zheng, Pu
    Gan, Haiyun
    Wang, Xuezheng
    Li, Longtu
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    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 stability2018Inngår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, nr 17, artikkel-id e99154Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 11. Li, Xiaoli
    et al.
    Jin, Xuejiao
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Liu, Xiaojing
    Zhang, Jiaxin
    Niu, Yanling
    Li, Jiani
    Li, Zhen
    Zhang, Jingjing
    Cao, Qinhong
    Hou, Wenya
    Du, Li-Lin
    Liu, Beidong
    Lou, Huiqiang
    Mck1 defines a key S-phase checkpoint effector in response to various degrees of replication threats2019Inngår i: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, nr 8, artikkel-id e1008136Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1 Delta to hydroxyurea (HU) reminiscent of mec1 Delta or rad53 Delta. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1 Delta dun1 Delta or mec1 Delta cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis. Author summary The appropriate amount and balance of four dNTPs are crucial for all cells correctly copying and passing on their genetic material generation by generation. Eukaryotes have developed an alert and response system to deal with the disturbance. Here, we uncovered a second-level effector branch. It is activated by the upstream surveillance kinase cascade, which can induce the expression of dNTP-producing enzymes. It can also reduce the inhibitor of these enzymes to further boost their activity according to the degrees of threats. These findings suggest a multi-level response system to guarantee the appropriate dNTP supply, which is essential to maintain genetic stability under various environmental challenges.

  • 12.
    Maicher, André
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gazy, Inbal
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Marjavaara, Lisette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Grinberg, Gilad
    Shemesh, Keren
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kupiec, Martin
    Rnr1, but not Rnr3, facilitates the sustained telomerase-dependent elongation of telomeres2017Inngår i: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 13, nr 10, artikkel-id e1007082Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 13.
    Mertz, Tony M
    et al.
    Omaha, Nebraska, USA.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Shcherbakova, Polina V
    Omaha, Nebraska, USA.
    Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity2015Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, nr 19, s. E2467-E2476Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 14. Nicholls, Thomas J.
    et al.
    Spåhr, Henrik
    Jiang, Shan
    Siira, Stefan J.
    Koolmeister, Camilla
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kauppila, Johanna H. K.
    Jiang, Min
    Kaever, Volkhard
    Rackham, Oliver
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Falkenberg, Maria
    Filipovska, Aleksandra
    Larsson, Nils-Göran
    Gustafsson, Claes M.
    Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria2019Inngår i: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 76, nr 5, s. 784-+Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.

  • 15. Ragu, Sandrine
    et al.
    Dardalhon, Michele
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iraqui, Ismail
    Buhagiar-Labarchede, Geraldine
    Grondin, Virginie
    Kienda, Guy
    Vernis, Laurence
    Chanet, Roland
    Kolodner, Richard D.
    Huang, Meng-Er
    Faye, Gerard
    Loss of the thioredoxin reductase Trr1 suppresses the genomic instability of peroxiredoxin tsa1 mutants2014Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 9, artikkel-id e108123Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1 Delta cells. In searching for spontaneous suppressors of synthetic lethality of tsa1 Delta rad51 Delta double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1 Delta mutant displayed a Can(R) mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1 Delta mutant reduced substantially the Can(R) mutation rate of tsa1 Delta strain (33-fold), and to a lesser extent, of rad51 Delta strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxidoreductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1 Delta -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1 Delta trx2 Delta cells, Yap1 was only moderately activated; consistently, the trx1 Delta trx2 Delta double deletion failed to efficiently rescue the viability of tsa1 Delta rad51 Delta. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1 Delta and trx1 Delta trx2 Delta strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.

  • 16. Schmidt, Tobias T
    et al.
    Reyes, Gloria
    Gries, Kerstin
    Ceylan, Cemile Ümran
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Meurer, Matthias
    Knop, Michael
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hombauer, Hans
    Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis2017Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 22, s. E4442-E4451Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 17. Schmidt, Tobias T.
    et al.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Reyes, Gloria X.
    Gries, Kerstin
    Gross, Maike
    Zhao, Boyu
    Yuan, Jui-Hung
    Wade, Rebecca
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hombauer, Hans
    A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance2019Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 1, s. 237-252Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 18.
    Sánchez, Arancha
    et al.
    Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA .
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    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 universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Russell, Paul
    Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA .
    Replication fork collapse and genome instability in dCMP deaminase mutant2012Inngår i: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 32, nr 21, s. 4445-4454Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 19.
    Tran, Phong
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wanrooij, Paulina H.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lorenzon, Paolo
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Anna Karin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anna-Karin
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Medini, Paolo
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    von Hofsten, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Stål, Per
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    De novo dNTP production is essential for normal postnatal murine heart development2019Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 394, nr 44, s. 15889-15897, artikkel-id jbc.RA119.009492Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in wild-type postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in wildtype hearts.

  • 20. van Mourik, Paula M
    et al.
    de Jong, Jannie
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kavšek, Alan
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Chang, Michael
    Upregulation of dNTP Levels After Telomerase Inactivation Influences Telomerase-Independent Telomere Maintenance Pathway Choice in Saccharomyces cerevisiae2018Inngår i: G3: Genes, Genomes, Genetics, ISSN 2160-1836, E-ISSN 2160-1836, Vol. 8, nr 8, s. 2551-2558Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 21. Xing, Xuanxuan
    et al.
    Kane, Daniel P.
    Bulock, Chelsea R.
    Moore, Elizabeth A.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Shcherbakova, Polina V.
    A recurrent cancer-associated substitution in DNA polymerase ε produces a hyperactive enzyme2019Inngår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, artikkel-id 374Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors. Severe mutator effects of the most common variant, Polε-P286R, modeled in yeast suggested that its pathogenicity involves yet unknown mechanisms beyond simple proofreading deficiency. We show that, despite producing a catastrophic amount of replication errors in vivo, the yeast Polε-P286R analog retains partial exonuclease activity and is more accurate than exonuclease-dead Polε. The major consequence of the arginine substitution is a dramatically increased DNA polymerase activity. This is manifested as a superior ability to copy synthetic and natural templates, extend mismatched primer termini, and bypass secondary DNA structures. We discuss a model wherein the cancer-associated substitution limits access of the 3'-terminus to the exonuclease site and promotes binding at the polymerase site, thus stimulating polymerization. We propose that the ultramutator effect results from increased polymerase activity amplifying the contribution of Polε errors to the genomic mutation rate.

  • 22. Yu, Chuanhe
    et al.
    Gan, Haiyun
    Serra-Cardona, Albert
    Zhang, Lin
    Gan, Songlin
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Johansson, Erik
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Xu, Rui-Ming
    Zhang, Zhiguo
    A mechanism for preventing asymmetric histone segregation onto replicating DNA strands2018Inngår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 361, nr 6409, s. 1386-+-, artikkel-id eaat8849Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging strand preference increases markedly in cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, due to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading vs lagging strands, and that Dbp3-Dpb4 plays a significant role in this poorly understood process.

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