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Chabes, Andrei, ProfessorORCID iD iconorcid.org/0000-0003-1708-8259
Biography [swe]

dNTPs and maintenance of genome stability

The four dNTPs (dATP, dTTP, dCTP, and dGTP) are the building blocks of DNA. A balanced supply and a correct overall concentration of dNTPs are key prerequisites for faithful genome replication. These requirements, along with the fact that the concentrations of dNTPs fluctuate during the cell cycle, mean that the production of dNTPs must be tightly regulated through multiple mechanisms.

We are investigating (i) how the genome integrity checkpoint regulates the concentration of dNTPs and how dNTPs regulate the activation of the genome integrity checkpoint, and (ii) how imbalanced dNTP pools affect the fidelity of replication and genome stability and how different replication errors are recognized and repaired. We hope to understand how metabolic changes in the dNTP pool balance affect aging, the development of cancer, and other genetic disorders.

Publications (10 of 70) Show all publications
Cerritelli, S. M., Iranzo, J., Sharma, S., Chabes, A., Crouch, R. J., Tollervey, D. & El Hage, A. (2020). High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase. Nucleic Acids Research, 48(8), 4274-4297
Open this publication in new window or tab >>High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase
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2020 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, no 8, p. 4274-4297Article in journal (Refereed) Published
Abstract [en]

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Place, publisher, year, edition, pages
Oxford Academic, 2020
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-170311 (URN)10.1093/nar/gkaa103 (DOI)32187369 (PubMedID)
Available from: 2020-05-02 Created: 2020-05-02 Last updated: 2020-05-05Bibliographically approved
Schmidt, T. T., Sharma, S., Reyes, G. X., Kolodziejczak, A., Wagner, T., Luke, B., . . . Hombauer, H. (2020). Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio. Nucleic Acids Research, 48(1), 264-277
Open this publication in new window or tab >>Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio
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2020 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, no 1, p. 264-277Article in journal (Refereed) Published
Abstract [en]

The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation. Here, we found that Met7-deficient strains show elevated mutation rates, but also increased levels of endogenous DNA damage resulting in gross chromosomal rearrangements (GCRs). Quantification of deoxyribonucleotide (dNTP) pools in cell extracts from met7Δ mutant revealed reductions in dTTP and dGTP that cause a constitutively active DNA damage checkpoint. In addition, we found that the absence of Met7 leads to dUTP accumulation, at levels that allowed its detection in yeast extracts for the first time. Consequently, a high dUTP/dTTP ratio promotes uracil incorporation into DNA, followed by futile repair cycles that compromise both mitochondrial and nuclear DNA integrity. In summary, this work highlights the importance of folate polyglutamylation in the maintenance of nucleotide homeostasis and genome stability.

Place, publisher, year, edition, pages
Oxford University Press, 2020
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-170313 (URN)10.1093/nar/gkz1006 (DOI)000524741700025 ()31647103 (PubMedID)
Available from: 2020-05-02 Created: 2020-05-02 Last updated: 2020-05-05Bibliographically approved
Forey, R., Poveda, A., Sharma, S., Barthe, A., Padioleau, I., Renard, C., . . . Pasero, P. (2020). Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication. Molecular Cell
Open this publication in new window or tab >>Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication
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2020 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164Article in journal (Refereed) Epub ahead of print
Abstract [en]

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
ATR, DNA replication, Mec1, S phase checkpoint, budding yeast, cell cycle, dNTP synthesis, fork collapse, mitotic catastrophe, replication timing
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-170312 (URN)10.1016/j.molcel.2020.02.021 (DOI)32169162 (PubMedID)
Available from: 2020-05-02 Created: 2020-05-02 Last updated: 2020-05-05
Davenne, T., Klintman, J., Sharma, S., Rigby, R. E., Blest, H. T. W., Cursi, C., . . . Rehwinkel, J. (2020). SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP.. Cell reports, 31(6), Article ID 107640.
Open this publication in new window or tab >>SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP.
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2020 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 31, no 6, article id 107640Article in journal (Refereed) Published
Abstract [en]

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

Keywords
BCX-1777, CyTOF, Immucillin H, SAMHD1, apoptosis, chronic lymphocytic leukemia, dGTP, dNTP, deoxyguanosine, forodesine
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-170758 (URN)10.1016/j.celrep.2020.107640 (DOI)32402273 (PubMedID)
Available from: 2020-05-14 Created: 2020-05-14 Last updated: 2020-05-15Bibliographically approved
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
Rentoft, M., Svensson, D., Sjödin, A., Olason, P. I., Sjöström, O., Nylander, C., . . . Johansson, E. (2019). A geographically matched control population efficiently limits the number of candidate disease-causing variants in an unbiased whole-genome analysis. PLoS ONE, 14(3), Article ID e0213350.
Open this publication in new window or tab >>A geographically matched control population efficiently limits the number of candidate disease-causing variants in an unbiased whole-genome analysis
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2019 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 3, article id e0213350Article in journal (Refereed) Published
Abstract [en]

Whole-genome sequencing is a promising approach for human autosomal dominant disease studies. However, the vast number of genetic variants observed by this method constitutes a challenge when trying to identify the causal variants. This is often handled by restricting disease studies to the most damaging variants, e.g. those found in coding regions, and overlooking the remaining genetic variation. Such a biased approach explains in part why the genetic causes of many families with dominantly inherited diseases, in spite of being included in whole-genome sequencing studies, are left unsolved today. Here we explore the use of a geographically matched control population to minimize the number of candidate disease-causing variants without excluding variants based on assumptions on genomic position or functional predictions. To exemplify the benefit of the geographically matched control population we apply a typical disease variant filtering strategy in a family with an autosomal dominant form of colorectal cancer. With the use of the geographically matched control population we end up with 26 candidate variants genome wide. This is in contrast to the tens of thousands of candidates left when only making use of available public variant datasets. The effect of the local control population is dual, it (1) reduces the total number of candidate variants shared between affected individuals, and more importantly (2) increases the rate by which the number of candidate variants are reduced as additional affected family members are included in the filtering strategy. We demonstrate that the application of a geographically matched control population effectively limits the number of candidate disease-causing variants and may provide the means by which variants suitable for functional studies are identified genome wide.

Place, publisher, year, edition, pages
Public Library of Science, 2019
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-158021 (URN)10.1371/journal.pone.0213350 (DOI)000462465800028 ()30917156 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2011.0042
Available from: 2019-04-10 Created: 2019-04-10 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
Chabes, A. (2019). dNTPs and maintenance of genome stability. FEBS Open Bio, 9, 21-21
Open this publication in new window or tab >>dNTPs and maintenance of genome stability
2019 (English)In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 9, p. 21-21Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
WILEY, 2019
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-164483 (URN)000486972400070 ()
Note

Supplement: 1. Meeting Abstract: S-10-3

Available from: 2019-10-22 Created: 2019-10-22 Last updated: 2019-10-22Bibliographically approved
Projects
dNTPs and maintenance of genome stability [2010-03552_VR]; Umeå UniversitydNTPs and maintenance of genome stability [2014-02262_VR]; Umeå UniversityAltered dNTP pools and genome instability [2018-02579_VR]; Umeå University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-1708-8259

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