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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 64) Show all publications
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, 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, article id jbc.RA119.009492Article in journal (Refereed) Epub ahead of print
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.

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)31300555 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2019-08-06
Wanrooij, P. H. & Chabes, A. (2019). Ribonucleotides in mitochondrial DNA. FEBS Letters
Open this publication in new window or tab >>Ribonucleotides in mitochondrial DNA
2019 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468Article in journal (Refereed) Epub ahead of print
Abstract [en]

The incorporation of ribonucleotides (rNMPs) into DNA during genome replication has gained substantial attention in recent years and has been shown to be a significant source of genomic instability. Studies in yeast and mammals have shown that the two genomes, the nuclear DNA (nDNA) and the mitochondrial DNA (mtDNA), differ with regard to their rNMP content. This is largely due to differences in rNMP repair - whereas rNMPs are efficiently removed from the nuclear genome, mitochondria lack robust mechanisms for removal of single rNMPs incorporated during DNA replication. In this minireview, we describe the processes that determine the frequency of rNMPs in the mitochondrial genome and summarise recent findings regarding the effect of incorporated rNMPs on mtDNA stability and function.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
Keywords
dNTP, genome stability, mitochondrial DNA, ribonucleotides
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-159088 (URN)10.1002/1873-3468.13440 (DOI)31093968 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Society for Medical Research (SSMF)
Available from: 2019-05-17 Created: 2019-05-17 Last updated: 2019-05-28
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
Peng, X. P., Lim, S., Li, S., Marjavaara, L., Chabes, A. & Zhao, X. (2018). Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites. PLoS Genetics, 14(1), Article ID e1007129.
Open this publication in new window or tab >>Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites
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2018 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 1, article id e1007129Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-144968 (URN)10.1371/journal.pgen.1007129 (DOI)000423718600006 ()29360860 (PubMedID)
Available from: 2018-02-21 Created: 2018-02-21 Last updated: 2018-06-09Bibliographically 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
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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