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Schmidt, T. T., Sharma, S., Reyes, G. X., Gries, K., Gross, M., Zhao, B., . . . Hombauer, H. (2019). A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance. Nucleic Acids Research, 47(1), 237-252
Open this publication in new window or tab >>A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance
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2019 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 1, p. 237-252Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Oxford University Press, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-154182 (URN)10.1093/nar/gky1154 (DOI)000462586700025 ()30462295 (PubMedID)
Funder
Swedish Cancer SocietyKnut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2018-12-13 Created: 2018-12-13 Last updated: 2019-04-12Bibliographically approved
Xing, X., Kane, D. P., Bulock, C. R., Moore, E. A., Sharma, S., Chabes, A. & Shcherbakova, P. V. (2019). A recurrent cancer-associated substitution in DNA polymerase ε produces a hyperactive enzyme. Nature Communications, 10, Article ID 374.
Open this publication in new window or tab >>A recurrent cancer-associated substitution in DNA polymerase ε produces a hyperactive enzyme
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2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 374Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-155817 (URN)10.1038/s41467-018-08145-2 (DOI)000456286400002 ()30670691 (PubMedID)
Funder
NIH (National Institute of Health), ES015869Swedish Cancer SocietySwedish Research Council
Available from: 2019-01-28 Created: 2019-01-28 Last updated: 2019-02-26Bibliographically approved
Tran, P., Wanrooij, P. H., Lorenzon, P., Sharma, S., Thelander, L., Nilsson, A. K., . . . Chabes, A. (2019). De novo dNTP production is essential for normal postnatal murine heart development. Journal of Biological Chemistry, 394(44), 15889-15897, Article ID jbc.RA119.009492.
Open this publication in new window or tab >>De novo dNTP production is essential for normal postnatal murine heart development
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2019 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 394, no 44, p. 15889-15897, article id jbc.RA119.009492Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2019
Keywords
cardiac function, cardiac muscle, dNTP metabolism, dNTP salvage, deoxyribonucleoside kinases, desmin, heart development, nucleoside/nucleotide biosynthesis, nucleoside/nucleotide metabolism, ribonucleotide reductase
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-161767 (URN)10.1074/jbc.RA119.009492 (DOI)000499478600002 ()31300555 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2020-01-03Bibliographically approved
Nicholls, T. J., Spåhr, H., Jiang, S., Siira, S. J., Koolmeister, C., Sharma, S., . . . Gustafsson, C. M. (2019). Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria. Molecular Cell, 76(5), 784-+
Open this publication in new window or tab >>Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria
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2019 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 76, no 5, p. 784-+Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2019
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-166839 (URN)10.1016/j.molcel.2019.09.010 (DOI)000500937100010 ()31588022 (PubMedID)
Funder
Swedish Research Council, 2015-00418Swedish Research Council, 2018-02439Swedish Research Council, 2018-02579Swedish Research Council, 2017-01257Swedish Cancer Society, 2016-816Swedish Cancer Society, 2016-599Swedish Cancer Society, 2017-631Swedish Cancer Society, 2018-602Knut and Alice Wallenberg Foundation, KAW 2017.0080Knut and Alice Wallenberg Foundation, KAW 2016.0050Wellcome trust, 213464/Z/18/Z
Available from: 2020-01-02 Created: 2020-01-02 Last updated: 2020-01-02Bibliographically approved
Li, X., Jin, X., Sharma, S., Liu, X., Zhang, J., Niu, Y., . . . Lou, H. (2019). Mck1 defines a key S-phase checkpoint effector in response to various degrees of replication threats. PLoS Genetics, 15(8), Article ID e1008136.
Open this publication in new window or tab >>Mck1 defines a key S-phase checkpoint effector in response to various degrees of replication threats
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2019 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, no 8, article id e1008136Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2019
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-164429 (URN)10.1371/journal.pgen.1008136 (DOI)000486222200029 ()31381575 (PubMedID)
Available from: 2019-10-22 Created: 2019-10-22 Last updated: 2019-10-22Bibliographically approved
Cohen, R., Milo, S., Sharma, S., Savidor, A. & Covo, S. (2019). Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress. DNA Repair, 83, Article ID 102674.
Open this publication in new window or tab >>Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress
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2019 (English)In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 83, article id 102674Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Ribonucleotide reductase, Fusarium oxysporum
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-165477 (URN)10.1016/j.dnarep.2019.102674 (DOI)000493214500005 ()31375409 (PubMedID)
Available from: 2019-12-04 Created: 2019-12-04 Last updated: 2019-12-04Bibliographically approved
Garbacz, M. A., Cox, P. B., Sharma, S., Lujan, S. A., Chabes, A. & Kunkel, T. A. (2019). The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity. Nucleic Acids Research, 47(8), 3986-3995
Open this publication in new window or tab >>The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity
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2019 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 8, p. 3986-3995Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Oxford University Press, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-156351 (URN)10.1093/nar/gkz048 (DOI)000473754300019 ()30698744 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2019-02-12 Created: 2019-02-12 Last updated: 2019-07-25Bibliographically approved
Yu, C., Gan, H., Serra-Cardona, A., Zhang, L., Gan, S., Sharma, S., . . . Zhang, Z. (2018). A mechanism for preventing asymmetric histone segregation onto replicating DNA strands. Science, 361(6409), 1386-+, Article ID eaat8849.
Open this publication in new window or tab >>A mechanism for preventing asymmetric histone segregation onto replicating DNA strands
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2018 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 361, no 6409, p. 1386-+-, article id eaat8849Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-150963 (URN)10.1126/science.aat8849 (DOI)000446142200050 ()30115745 (PubMedID)
Funder
NIH (National Institute of Health), R35GM118015Swedish Cancer SocietySwedish Research Council
Note

Special Issue: SI

Available from: 2018-08-21 Created: 2018-08-21 Last updated: 2018-10-31Bibliographically approved
Bacal, J., Moriel-Carretero, M., Pardo, B., Barthe, A., Sharma, S., Chabes, A., . . . Pasero, P. (2018). Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage. EMBO Journal, 37(21), Article ID e99319.
Open this publication in new window or tab >>Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage
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2018 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 21, article id e99319Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018
Keywords
DNA replication, S‐phase checkpoint, elongation, initiation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-151327 (URN)10.15252/embj.201899319 (DOI)000449944900003 ()30158111 (PubMedID)
Available from: 2018-08-31 Created: 2018-08-31 Last updated: 2019-01-08Bibliographically approved
Li, S., Xu, Z., Xu, J., Zuo, L., Yu, C., Zheng, P., . . . Li, Q. (2018). Rtt105 functions as a chaperone for replication protein A to preserve genome stability. EMBO Journal, 37(17), Article ID e99154.
Open this publication in new window or tab >>Rtt105 functions as a chaperone for replication protein A to preserve genome stability
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2018 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 17, article id e99154Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018
Keywords
RPA chaperone, Rtt105, replication fork, replication stress
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-150962 (URN)10.15252/embj.201899154 (DOI)000443413000009 ()30065069 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietyKnut and Alice Wallenberg Foundation
Available from: 2018-08-21 Created: 2018-08-21 Last updated: 2018-09-21Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-2713-5813

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