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Publications (10 of 55) Show all publications
Lawir, D.-F., Soza-Ried, C., Iwanami, N., Siamishi, I., Bylund, G., O´Meara, C., . . . Boehm, T. (2024). Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish. Communications Biology, 7(1), Article ID 31.
Open this publication in new window or tab >>Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish
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2024 (English)In: Communications Biology, E-ISSN 2399-3642, Vol. 7, no 1, article id 31Article in journal (Refereed) Published
Abstract [en]

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Place, publisher, year, edition, pages
Nature Publishing Group, 2024
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-219482 (URN)10.1038/s42003-023-05692-3 (DOI)38182651 (PubMedID)2-s2.0-85181464404 (Scopus ID)
Funder
Max Planck SocietyEU, European Research Council, 323126
Note

Author correction: Lawir, DF., Soza-Ried, C., Iwanami, N. et al. Author Correction: Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish. Commun Biol 7, 247 (2024). DOI: 10.1038/s42003-024-05899-y

Available from: 2024-01-24 Created: 2024-01-24 Last updated: 2024-07-02Bibliographically approved
Parkash, V., Kulkarni, Y., Bylund, G. O., Osterman, P., Kamerlin, S. C. & Johansson, E. (2023). A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides. Nucleic Acids Research, 51(20), 11225-11238
Open this publication in new window or tab >>A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides
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2023 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 20, p. 11225-11238Article in journal (Refereed) Published
Abstract [en]

The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2′-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.

Place, publisher, year, edition, pages
Oxford University Press, 2023
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-218101 (URN)10.1093/nar/gkad817 (DOI)37819038 (PubMedID)2-s2.0-85178042069 (Scopus ID)
Funder
Vinnova, 2018-04969Swedish Research Council Formas, 2019-02496Swedish Cancer Society, 2018-05973Swedish Research Council, 2018-07152Swedish Research Council, 2019-03499Swedish Research Council, 2021-01104
Available from: 2023-12-15 Created: 2023-12-15 Last updated: 2024-07-02Bibliographically approved
Barbari, S. R., Beach, A. K., Markgren, J. G., Parkash, V., Moore, E. A., Johansson, E. & Shcherbakova, P. V. (2022). Enhanced polymerase activity permits efficient synthesis by cancer-Associated DNA polymerase variants at low dNTP levels. Nucleic Acids Research, 50(14), 8023-8040
Open this publication in new window or tab >>Enhanced polymerase activity permits efficient synthesis by cancer-Associated DNA polymerase variants at low dNTP levels
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2022 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 14, p. 8023-8040Article in journal (Refereed) Published
Abstract [en]

Amino acid substitutions in the exonuclease domain of DNA polymerase (Pol) cause ultramutated tumors. Studies in model organisms suggested pathogenic mechanisms distinct from a simple loss of exonuclease. These mechanisms remain unclear for most recurrent Pol mutations. Particularly, the highly prevalent V411L variant remained a long-standing puzzle with no detectable mutator effect in yeast despite the unequivocal association with ultramutation in cancers. Using purified four-subunit yeast Pol, we assessed the consequences of substitutions mimicking human V411L, S459F, F367S, L424V and D275V. While the effects on exonuclease activity vary widely, all common cancer-Associated variants have increased DNA polymerase activity. Notably, the analog of Pol-V411L is among the strongest polymerases, and structural analysis suggests defective polymerase-To-exonuclease site switching. We further show that the V411L analog produces a robust mutator phenotype in strains that lack mismatch repair, indicating a high rate of replication errors. Lastly, unlike wild-Type and exonuclease-dead Pol, hyperactive variants efficiently synthesize DNA at low dNTP concentrations. We propose that this characteristic could promote cancer cell survival and preferential participation of mutator polymerases in replication during metabolic stress. Our results support the notion that polymerase fitness, rather than low fidelity alone, is an important determinant of variant pathogenicity.

Place, publisher, year, edition, pages
Oxford University Press, 2022
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-199009 (URN)10.1093/nar/gkac602 (DOI)000823814600001 ()35822874 (PubMedID)2-s2.0-85136314031 (Scopus ID)
Funder
Swedish Cancer SocietySwedish Research Council, 2018-07152Vinnova, 2018-04969Swedish Research Council Formas, 2019-02496
Available from: 2022-09-02 Created: 2022-09-02 Last updated: 2024-07-02Bibliographically approved
Posse, V., Johansson, E. & Diffley, J. F. X. (2021). Eukaryotic DNA replication with purified budding yeast proteins. In: Brandt F. Eichman (Ed.), The DNA replication-repair interface: (pp. 1-33). Elsevier, 661
Open this publication in new window or tab >>Eukaryotic DNA replication with purified budding yeast proteins
2021 (English)In: The DNA replication-repair interface / [ed] Brandt F. Eichman, Elsevier, 2021, Vol. 661, p. 1-33Chapter in book (Refereed)
Abstract [en]

The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.

Place, publisher, year, edition, pages
Elsevier, 2021
Series
Methods in Enzymology, ISSN 0076-6879 ; 661
Keywords
Protein expression, Chromosome duplication, Protein purification, DNA replication, DNA replication assay, DNA replication in vitro, Reconstituted yeast DNA replication
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-190683 (URN)10.1016/bs.mie.2021.08.018 (DOI)000750885400002 ()34776208 (PubMedID)2-s2.0-85117767031 (Scopus ID)9780323907330 (ISBN)
Available from: 2021-12-21 Created: 2021-12-21 Last updated: 2024-07-02Bibliographically approved
Pinto, M. N., ter Beek, J., Ekanger, L. A., Johansson, E. & Barton, J. K. (2021). The [4Fe4S] Cluster of Yeast DNA Polymerase ϵ Is Redox Active and Can Undergo DNA-Mediated Signaling. Journal of the American Chemical Society, 143(39), 16147-16153
Open this publication in new window or tab >>The [4Fe4S] Cluster of Yeast DNA Polymerase ϵ Is Redox Active and Can Undergo DNA-Mediated Signaling
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2021 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 143, no 39, p. 16147-16153Article in journal (Refereed) Published
Abstract [en]

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo, was used to limit DNA degradation. While no redox signal is apparent for Pol2COREexo on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2COREexo, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]2+, to oxidized Pol2COREexo bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIIIY82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2COREexo. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2COREexo via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2021
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-189183 (URN)10.1021/jacs.1c07150 (DOI)000706193200025 ()34559527 (PubMedID)2-s2.0-85116594622 (Scopus ID)
Available from: 2021-11-12 Created: 2021-11-12 Last updated: 2024-07-02Bibliographically approved
Johansson, E. & Diffley, J. F. (2021). Unchecked nick ligation can promote localized genome re-replication. Current Biology, 31(11), R710-R711
Open this publication in new window or tab >>Unchecked nick ligation can promote localized genome re-replication
2021 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 31, no 11, p. R710-R711Article in journal (Refereed) Published
Abstract [en]

Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a collapsed replication fork comprising a single-ended double-strand break and a 'hybrid nick' with parental DNA on one side and nascent DNA on the other (Figure 1A). We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a fork from an adjacent origin can promote localized re-replication if the hybrid nick is ligated. We have modelled this situation with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.

Place, publisher, year, edition, pages
Elsevier, 2021
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-186360 (URN)10.1016/j.cub.2021.03.043 (DOI)000658932200005 ()34102115 (PubMedID)2-s2.0-85107917996 (Scopus ID)
Available from: 2021-07-23 Created: 2021-07-23 Last updated: 2024-07-02Bibliographically approved
Svensson, D., Rentoft, M., Dahlin, A. M., Lundholm, E., Olason, P. I., Sjödin, A., . . . Johansson, E. (2020). A whole-genome sequenced control population in northern Sweden reveals subregional genetic differences. PLOS ONE, 15(9), Article ID e0237721.
Open this publication in new window or tab >>A whole-genome sequenced control population in northern Sweden reveals subregional genetic differences
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2020 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 15, no 9, article id e0237721Article in journal (Refereed) Published
Abstract [en]

The number of national reference populations that are whole-genome sequenced are rapidly increasing. Partly driving this development is the fact that genetic disease studies benefit from knowing the genetic variation typical for the geographical area of interest. A whole-genome sequenced Swedish national reference population (n = 1000) has been recently published but with few samples from northern Sweden. In the present study we have whole-genome sequenced a control population (n = 300) (ACpop) from Västerbotten County, a sparsely populated region in northern Sweden previously shown to be genetically different from southern Sweden. The aggregated variant frequencies within ACpop are publicly available (DOI 10.17044/NBIS/G000005) to function as a basic resource in clinical genetics and for genetic studies. Our analysis of ACpop, representing approximately 0.11% of the population in Västerbotten, indicates the presence of a genetic substructure within the county. Furthermore, a demographic analysis showed that the population from which samples were drawn was to a large extent geographically stationary, a finding that was corroborated in the genetic analysis down to the level of municipalities. Including ACpop in the reference population when imputing unknown variants in a Västerbotten cohort resulted in a strong increase in the number of high-confidence imputed variants (up to 81% for variants with minor allele frequency < 5%). ACpop was initially designed for cancer disease studies, but the genetic structure within the cohort will be of general interest for all genetic disease studies in northern Sweden.

Place, publisher, year, edition, pages
Public Library Science, 2020
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-175837 (URN)10.1371/journal.pone.0237721 (DOI)000571887500123 ()32915809 (PubMedID)2-s2.0-85090917774 (Scopus ID)
Available from: 2020-10-16 Created: 2020-10-16 Last updated: 2023-03-23Bibliographically 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, 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)2-s2.0-85063572524 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, 2011.0042
Available from: 2019-04-10 Created: 2019-04-10 Last updated: 2024-07-02Bibliographically approved
Parkash, V., Kulkarni, Y., ter Beek, J., Shcherbakova, P. V., Kamerlin, S. C. & Johansson, E. (2019). Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon. Nature Communications, 10, Article ID 373.
Open this publication in new window or tab >>Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon
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2019 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 10, article id 373Article in journal (Refereed) Published
Abstract [en]

The most frequently recurring cancer-associated DNA polymerase epsilon (Pol epsilon) mutation is a P286R substitution in the exonuclease domain. While originally proposed to increase genome instability by disrupting exonucleolytic proofreading, the P286R variant was later found to be significantly more pathogenic than Pol epsilon proofreading deficiency per se. The mechanisms underlying its stronger impact remained unclear. Here we report the crystal structure of the yeast orthologue, Pol epsilon-P301R, complexed with DNA and an incoming dNTP. Structural changes in the protein are confined to the exonuclease domain, with R301 pointing towards the exonuclease site. Molecular dynamics simulations suggest that R301 interferes with DNA binding to the exonuclease site, an outcome not observed with the exonuclease-inactive Pol epsilon-D290A, E292A variant lacking the catalytic residues. These results reveal a distinct mechanism of exonuclease inactivation by the P301R substitution and a likely basis for its dramatically higher mutagenic and tumorigenic effects.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-156315 (URN)10.1038/s41467-018-08114-9 (DOI)000456286400001 ()30670696 (PubMedID)2-s2.0-85060366876 (Scopus ID)
Available from: 2019-02-21 Created: 2019-02-21 Last updated: 2024-07-02Bibliographically approved
ter Beek, J., Parkash, V., Bylund, G., Osterman, P., Sauer-Eriksson, A. E. & Johansson, E. (2019). Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ. Nucleic Acids Research, 47(11), 5712-5722
Open this publication in new window or tab >>Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ
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2019 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 11, p. 5712-5722Article in journal (Refereed) Published
Abstract [en]

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2CORE (with the CysX cysteine motif) are likely to coordinate an Fe–S cluster. Here, we present two new crystal structures of Pol2CORE with an Fe–S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysAMUT (C2111S/C2133S), and Pol ϵ CysBMUT (C2167S/C2181S) all have an Fe–S cluster that is not present in Pol ϵ CysXMUT (C665S/C668S). Pol ϵ CysAMUT and Pol ϵ CysBMUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysXMUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysXMUT are inviable. In conclusion, Pol ϵ has a single Fe–S cluster bound at the base of the P-domain, and this Fe–S cluster is essential for cell viability and polymerase activity.

Place, publisher, year, edition, pages
Oxford University Press, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-161925 (URN)10.1093/nar/gkz248 (DOI)000475702000027 ()30968138 (PubMedID)2-s2.0-85068487970 (Scopus ID)
Available from: 2019-08-06 Created: 2019-08-06 Last updated: 2019-08-06Bibliographically approved
Projects
Symposium on Enzymes in Nucleic Acid synthesis [2008-01968_VR]; Umeå UniversityInvestigation of a novel function of human DNA Polymerase theta (POLQ) [2010-03558_VR]; Umeå UniversityStructural and functional studies of DNA polymerase epsilon [2010-05071_VR]; Umeå UniversityStructural and functional studies of DNA polymerase epsilon [2013-05888_VR]; Umeå UniversityThe mechanism of eukaryotic leading-strand DNA synthesis [2017-04096_VR]; Umeå University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-8526-6224

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