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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 61) Show all publications
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(1), 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, no 1, 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.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-155817 (URN)10.1038/s41467-018-08145-2 (DOI)30670691 (PubMedID)
Available from: 2019-01-28 Created: 2019-01-28 Last updated: 2019-01-28
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
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 1362-4962Article in journal (Refereed) Epub ahead of print
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.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-156351 (URN)10.1093/nar/gkz048 (DOI)30698744 (PubMedID)
Available from: 2019-02-12 Created: 2019-02-12 Last updated: 2019-02-12
Schmidt, T. T., Sharma, S., Reyes, G. X., Gries, K., Gross, M., Zhao, B., . . . Hombauer, H. (2018). A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance. Nucleic Acids Research
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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2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed) Epub ahead of print
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, 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-154182 (URN)10.1093/nar/gky1154 (DOI)30462295 (PubMedID)
Available from: 2018-12-13 Created: 2018-12-13 Last updated: 2018-12-14
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
Coquel, F., Silva, M.-J., Técher, H., Zadorozhny, K., Sharma, S., Nieminuszczy, J., . . . Pasero, P. (2018). SAMHD1 acts at stalled replication forks to prevent interferon induction. Nature, 557(7703), 57-61
Open this publication in new window or tab >>SAMHD1 acts at stalled replication forks to prevent interferon induction
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2018 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 557, no 7703, p. 57-61Article in journal (Refereed) Published
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.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-147779 (URN)10.1038/s41586-018-0050-1 (DOI)000431234500029 ()29670289 (PubMedID)
Available from: 2018-05-17 Created: 2018-05-17 Last updated: 2018-06-09Bibliographically approved
Lanz, M. C., Oberly, S., Sanford, E. J., Sharma, S., Chabes, A. & Smolka, M. B. (2018). Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling. Genes & Development, 32(11-12), 822-835
Open this publication in new window or tab >>Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
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2018 (English)In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 32, no 11-12, p. 822-835Article in journal (Refereed) Published
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.

Keywords
ATR, DNA replication, Dna2, Dpb11, Mec1, gross chromosomal rearrangements
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-150961 (URN)10.1101/gad.308148.117 (DOI)000436070000008 ()29899143 (PubMedID)2-s2.0-85048778293 (Scopus ID)
Available from: 2018-08-21 Created: 2018-08-21 Last updated: 2018-08-31Bibliographically approved
Kong, Z., Jia, S., Chabes, A. L., Appelblad, P., Lundmark, R., Moritz, T. & Chabes, A. (2018). Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Nucleic Acids Research, 46(11), Article ID e66.
Open this publication in new window or tab >>Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry
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2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 11, article id e66Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
Oxford University Press, 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-145936 (URN)10.1093/nar/gky203 (DOI)000438362400003 ()29554314 (PubMedID)
Funder
Swedish Research Council
Available from: 2018-03-22 Created: 2018-03-22 Last updated: 2018-09-28Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-1708-8259

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