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  • 1.
    Aksenova, Anna
    et al.
    Department of Biology, Tufts University, Medford, Massachusetts, United States of America.
    Volkov, Kirill
    School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.
    Maceluch, Jaroslaw
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pursell, Zachary F
    Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America.
    Rogozin, Igor B
    National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America.
    Kunkel, Thomas A
    Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Heath, Department of Health and Human Services, Research Triangle Park, North Carolina, United States of America.
    Pavlov, Youri I
    Eppley Institute for Research in Cancer, Department of Biochemistry and Molecular Biology, and Department of Microbiology and Pathology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε2010In: PLoS genetics, ISSN 1553-7404, Vol. 6, no 11, p. e1001209-Article in journal (Refereed)
    Abstract [en]

    Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.

  • 2. Asturias, Francisco J
    et al.
    Cheung, Iris K
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Chilkova, Olga
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wepplo, Daniel
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy.2006In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 13, no 1, p. 35-43Article in journal (Refereed)
  • 3.
    Barbari, Stephanie R.
    et al.
    Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE, Omaha, United States.
    Beach, Annette K.
    Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE, Omaha, United States.
    Markgren, Joel G.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Parkash, Vimal
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Moore, Elizabeth A.
    Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE, Omaha, United States.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shcherbakova, Polina V
    Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE, Omaha, United States.
    Enhanced polymerase activity permits efficient synthesis by cancer-Associated DNA polymerase variants at low dNTP levels2022In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 14, p. 8023-8040Article in journal (Refereed)
    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.

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  • 4.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Hjortsberg, K
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase.1993In: Proceedings of the National Academy of Science U S A, ISSN 0027-8424, Vol. 90, no 23, p. 11322-6Article in journal (Refereed)
  • 5.
    Chilkova, Olga
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Jonsson, Bengt-Harald
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae.2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 16, p. 14082-14086Article in journal (Refereed)
  • 6.
    Chilkova, Olga
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Stenlund, Peter
    Isoz, Isabelle
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Stith, Carrie M
    Grabowski, Pawel
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA.2007In: Nucleic Acids Research, ISSN 1362-4962, Vol. 35, no 19, p. 6588-6597Article in journal (Refereed)
    Abstract [en]

    Saccharomyces cerevisiae DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol delta and Pol epsilon in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol epsilon has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol delta has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol delta and Pol epsilon was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol delta and Pol epsilon on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol epsilon when compared to Pol delta. We conclude that Pol epsilon and Pol delta exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.

  • 7. Filatov, D
    et al.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Induction of the mouse ribonucleotide reductase R1 and R2 genes in response to DNA damage by UV light.1996In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 271, no 39, p. 23698-704Article in journal (Refereed)
  • 8. Fortune, John M
    et al.
    Pavlov, Youri I
    Welch, Carrie M
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M J
    Kunkel, Thomas A
    Saccharomyces cerevisiae DNA polymerase delta: high fidelity for base substitutions but lower fidelity for single- and multi-base deletions.2005In: Journal of biological chemistry, ISSN 0021-9258, Vol. 280, no 33, p. 29980-7Article in journal (Refereed)
  • 9.
    Ganai, Rais A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Howard Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, USA.
    Zhang, Xiao-Ping
    Heyer, Wolf-Dietrich
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Strand displacement synthesis by yeast DNA polymerase epsilon2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 17, p. 8229-8240Article in journal (Refereed)
    Abstract [en]

    DNA polymerase epsilon (Pol epsilon) is a replicative DNA polymerase with an associated 3'aEuro"5' exonuclease activity. Here, we explored the capacity of Pol epsilon to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol epsilon is unable to carry out extended strand displacement synthesis unless its 3'aEuro"5' exonuclease activity is removed. However, the wild-type Pol epsilon holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol epsilon from synthesizing DNA up to the fork junction. This inhibition was observed for Pol epsilon but not with Pol delta, RB69 gp43 or Pol eta. Neither was Pol epsilon able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol epsilon is distributive. Our results suggest that Pol epsilon is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol epsilon participates in short-patch base excision repair and ribonucleotide excision repair.

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  • 10.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Switching between polymerase and exonuclease sites in DNA polymerase ε2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 2, p. 932-942Article in journal (Refereed)
    Abstract [en]

    The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.

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  • 11.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Howard Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NY 10016, USA.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA Replication - A Matter of Fidelity2016In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 62, no 5, p. 745-755Article, review/survey (Refereed)
    Abstract [en]

    The fidelity of DNA replication is determined by many factors, here simplified as the contribution of the DNA polymerase (nucleotide selectivity and proofreading), mismatch repair, a balanced supply of nucleotides, and the condition of the DNA template (both in terms of sequence context and the presence of DNA lesions). This review discusses the contribution and interplay between these factors to the overall fidelity of DNA replication.

  • 12.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University.
    Modulation of strand displacement synthesis of DNA polymerase ε byprocessive 3'- 5' exonuclease activity.Manuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    DNA polymerase epilson (Pol ε) is a replicative DNA polymerase with a processive 3'-5' exonuclease activity. Here we ask if Pol ε is capable of performing strand displacement synthesis, which influence many DNA processes in vivo. We found that Polε is unable to carry out extended strand displacement synthesis unless the proofreading is inactivated. However, Pol ε efficiently displaced one nucleotide when encountering double stranded DNA after filling a gap of 8 nucleotides. An abasic moiety at the 5'-end of the downstream primer was as efficiently displaced and still only with one nucleotide. Pol ε also efficiently recognized the 3'-OH innicked DNA and displaced the 5'- nucleotide, regardless if it was a normal phosphorylated deoxyribonucleotide or a ribonucleotide. A flap, mimicking a D-loop or a hairpin structure, on the 5'-end of the blocking primer inhibited Pol ε, and did not allow Pol ε to efficiently synthesize DNA up to the junction with double-stranded DNA. Finally, we show that strand displacement synthesis is limited by the processive 3'–5' exonuclease activity in Pol ε. Our results suggests that Pol ε is unable to extend D-loops during homologous recombination or participate in long-patch base excision repair based on the inhibition by the 5'–flap of the downstream primer. Our results do, however, support that Pol ε may participate in short patch base excision repair and ribonucleotide excision repair.

  • 13.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast DNA Polymerase epsilon Catalytic Core and Holoenzyme Have Comparable Catalytic Rates2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3825-3835Article in journal (Refereed)
    Abstract [en]

    The holoenzyme of yeast DNApolymerase ε (Pol ε) consists of four subunits– Pol2, Dpb2, Dpb3, and Dpb4. A proteasesensitivesite results in a N-terminalproteolytic fragment of Pol2, called Pol2core,that consists of the catalytic core of Pol ε andretains both polymerase and exonucleaseactivities. Pre-steady-state kinetics showedthat the exonuclease rates on single-stranded,double-stranded, and mismatched DNA werecomparable between Pol ε and Pol2core. Singleturnover pre-steady-state kinetics alsoshowed that the kpol of Pol ε and Pol2core werecomparable when pre-loading the polymeraseonto the primer-template before adding Mg2+and dTTP. However, a global fit of the dataover six sequential nucleotide incorporationsrevealed that the overall polymerization rateand processivity was higher for Pol ε than forPol2core. The largest difference was observedwhen challenged for the formation of aternary complex and incorporation of thefirst nucleotide. Pol ε needed less than asecond to incorporate a nucleotide, butseveral seconds passed before Pol2coreincorporated detectable levels of the firstnucleotide. We conclude that the accessorysubunits and the C-terminus of Pol2 do notinfluence the catalytic rate of Pol ε butfacilitate the loading and incorporation of thefirst nucleotide by Pol ε.

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  • 14. Garg, Parie
    et al.
    Stith, Carrie M
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M
    Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication.2004In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 18, no 22, p. 2764-2773Article in journal (Refereed)
  • 15. Haracska, L
    et al.
    Unk, I
    Johnson, R E
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, P M
    Prakash, S
    Prakash, L
    Roles of yeast DNA polymerases delta and zeta and of Rev1 in the bypass of abasic sites.2001In: Genes & Development, ISSN 0890-9369, Vol. 15, no 8, p. 945-54Article in journal (Refereed)
  • 16.
    Hogg, Matthew
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA Polymerase ε2012In: The eukaryotic replisome: a guide to protein structure and function / [ed] MacNeill S, Springer Science+Business Media B.V., 2012, Vol. 62, p. 237-57Chapter in book (Refereed)
    Abstract [en]

    DNA polymerase ε (Pol ε) is one of three replicative DNA polymerases in eukaryotic cells. Pol ε is a multi-subunit DNA polymerase with many functions. For example, recent studies in yeast have suggested that Pol ε is essential during the initiation of DNA replication and also participates during leading strand synthesis. In this chapter, we will discuss the structure of Pol ε, the individual subunits and their function.

  • 17.
    Hogg, Matthew
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ganai, Rais Ahmad
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural basis for processive DNA synthesis by yeast DNA polymerase ε2014In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 21, no 1, p. 49-56Article in journal (Refereed)
    Abstract [en]

    DNA polymerase ε (Pol ε) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ε (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ε has the highest fidelity among B-family polymerases despite the absence of an extended b-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ε to encircle the nascent doublestranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes

  • 18.
    Hogg, Matthew
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Promiscuous DNA synthesis by human DNA polymerase θ2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 6, p. 2611-2622Article in journal (Refereed)
    Abstract [en]

    The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3'-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks.

  • 19.
    Isoz, Isabelle
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Persson, Ulf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Volkov, Kirill
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 22, p. 11545-11553Article in journal (Refereed)
    Abstract [en]

    DNA polymerase ε (Pol ε) participates in the synthesis of the leading strand during DNA replication in Saccharomyces cerevisiae. Pol ε comprises four subunits: the catalytic subunit, Pol2, and three accessory subunits, Dpb2, Dpb3 and Dpb4. DPB2 is an essential gene with unclear function. A genetic screen was performed in S. cerevisiae to isolate lethal mutations in DPB2. The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation. This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits. This resulted in the purification of a Pol2/Dpb3/Dpb4 complex that possessed high specific activity and high processivity and holoenzyme assays with PCNA, RFC and RPA on a single-primed circular template did not reveal any defects in replication efficiency. In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity. Thus, the C-terminal motif of Dpb2 that we have identified may instead be required for Dpb2 to fulfill an essential structural role at the replication origin or at the replication fork.

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  • 20.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA och cancerutveckling2013In: Cancerforskning på nya vägar: en bok från Froskningens dag 2013, Medicinska fakulteten vid Umeå universitet / [ed] Mattias Grundström Mitz och Lena Åminne, Umeå: Umeå universitet , 2013, 1, p. 15-22Chapter in book (Other (popular science, discussion, etc.))
  • 21.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gene structure and regulation of the expression of the R1 and R2 subunits of mouse ribonucleotide reductase.1994In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 370, p. 721-4Article in journal (Refereed)
  • 22.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Chromosome Replication Laboratory, The Francis Crick Institute, London, UK.
    Diffley, John F X
    Chromosome Replication Laboratory, Francis Crick Institute, London, United Kingdom.
    Unchecked nick ligation can promote localized genome re-replication2021In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 31, no 11, p. R710-R711Article in journal (Refereed)
    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.

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  • 23.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Dixon, Nicholas
    Replicative DNA polymerases2013In: Cold Spring Harbor Perspectives in Biology, E-ISSN 1943-0264, Vol. 5, no 6, p. a012799-Article in journal (Refereed)
    Abstract [en]

    In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact.

  • 24.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Garg, Parie
    Burgers, Peter M J
    The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding.2004In: Journal of biological chemistry, ISSN 0021-9258, Vol. 279, no 3, p. 1907-15Article in journal (Refereed)
  • 25.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Hjortsberg, K
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Two YY-1-binding proximal elements regulate the promoter strength of the TATA-less mouse ribonucleotide reductase R1 gene.1998In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 273, no 45, p. 29816-21Article in journal (Refereed)
  • 26.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    MacNeill, Stuart A
    The eukaryotic replicative DNA polymerases take shape.2010In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 35, no 6, p. 339-347Article, review/survey (Refereed)
    Abstract [en]

    Three multi-subunit DNA polymerase enzymes lie at the heart of the chromosome replication machinery in the eukaryotic cell nucleus. Through a combination of genetic, molecular biological and biochemical analysis, significant advances have been made in understanding the essential roles played by each of these enzymes at the replication fork. Until very recently, however, little information was available on their three-dimensional structures. Lately, a series of crystallographic and electron microscopic studies has been published, allowing the structures of the complexes and their constituent subunits to be visualised in detail for the first time. Taken together, these studies provide significant insights into the molecular makeup of the replication machinery in eukaryotic cells and highlight a number of key areas for future investigation.

  • 27.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Majka, J
    Burgers, P M
    Structure of DNA polymerase delta from Saccharomyces cerevisiae.2001In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 276, no 47, p. 43824-8Article in journal (Refereed)
  • 28.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Skogman, E
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    The TATA-less promoter of mouse ribonucleotide reductase R1 gene contains a TFII-I binding initiator element essential for cell cycle-regulated transcription.1995In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 270, no 50, p. 30162-7Article in journal (Other academic)
  • 29.
    Johansson, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Speck, Christian
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    A top-down view on DNA replication and recombination from 9,000 feet above sea level2011In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 12, no 4, article id 304Article in journal (Refereed)
    Abstract [en]

    A report of the Keystone Symposium 'DNA Replication and Recombination' held in Keystone, USA, 27 February to 4 March 2011.

  • 30. Kamath-Loeb, A S
    et al.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, P M
    Loeb, L A
    Functional interaction between the Werner Syndrome protein and DNA polymerase delta.2000In: Proceedings or the National Academy of Sciences U S A, ISSN 0027-8424, Vol. 97, no 9, p. 4603-8Article in journal (Refereed)
  • 31. Kamath-Loeb, A S
    et al.
    Loeb, L A
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, P M
    Fry, M
    Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence.2001In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 276, no 19, p. 16439-46Article in journal (Refereed)
  • 32.
    Lawir, Divine-Fondzenyuy
    et al.
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Soza-Ried, Cristian
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Iwanami, Norimasa
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Siamishi, Iliana
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    O´Meara, Connor
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Sikora, Katarzyna
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; Bioinformatic Unit, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Kanzler, Benoît
    Transgenic Mouse Core Facility, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schorpp, Michael
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Cauchy, Pierre
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.
    Boehm, Thomas
    Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; Institute for Immunodeficiency, Center for Chronic Immunodeficiency (CCI), University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
    Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish2024In: Communications Biology, E-ISSN 2399-3642, Vol. 7, no 1, article id 31Article in journal (Refereed)
    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.

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  • 33. McCulloch, Scott D
    et al.
    Kokoska, Robert J
    Chilkova, Olga
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Welch, Carrie M
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M J
    Kunkel, Thomas A
    Enzymatic switching for efficient and accurate translesion DNA replication.2004In: Nucleic Acids Research, ISSN 1362-4962, Vol. 32, no 15, p. 4665-75Article in journal (Refereed)
  • 34. Nick McElhinny, Stephanie A
    et al.
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Clark, Alan B
    Watt, Danielle L
    Watts, Brian E
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kunkel, Thomas A
    Genome instability due to ribonucleotide incorporation into DNA2010In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 6, no 10, p. 774-81Article in journal (Refereed)
    Abstract [en]

    Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase ϵ (Pol ϵ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol ϵ compared to wild-type Pol ϵ. This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S phase, had elevated dNTP pools and generated 2-5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

  • 35. Nick McElhinny, Stephanie A
    et al.
    Watts, Brian E
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Watt, Danielle L
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M J
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kunkel, Thomas A
    Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 11, p. 4949-4954Article in journal (Refereed)
    Abstract [en]

    Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36- to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase epsilon, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol delta and Pol alpha, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols alpha, delta, and epsilon, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase epsilon has difficulty bypassing a single rNMP present within a DNA template.

  • 36.
    Parkash, Vimal
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kulkarni, Yashraj
    Department of Chemistry - BMC, Uppsala University, Box 576, Uppsala, Sweden; Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
    Bylund, Göran O.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kamerlin, Shina Caroline Lynn
    Department of Chemistry - BMC, Uppsala University, Box 576, Uppsala, Sweden; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, GA, Atlanta, United States.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 20, p. 11225-11238Article in journal (Refereed)
    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.

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  • 37.
    Parkash, Vimal
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kulkarni, Yashraj
    ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shcherbakova, Polina V.
    Kamerlin, Shina Caroline Lynn
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon2019In: Nature Communications, E-ISSN 2041-1723, Vol. 10, article id 373Article in journal (Refereed)
    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.

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  • 38.
    Pinto, Miguel N.
    et al.
    Division of Chemistry and Chemical Engineering, California Institute of Technology, CA, Pasadena, United States.
    ter Beek, Josy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ekanger, Levi A.
    Division of Chemistry and Chemical Engineering, California Institute of Technology, CA, Pasadena, United States; Department of Chemistry, The College of New Jersey, NJ, Ewing, United States.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Barton, Jacqueline K.
    Division of Chemistry and Chemical Engineering, California Institute of Technology, CA, Pasadena, United States.
    The [4Fe4S] Cluster of Yeast DNA Polymerase ϵ Is Redox Active and Can Undergo DNA-Mediated Signaling2021In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 143, no 39, p. 16147-16153Article in journal (Refereed)
    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.

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  • 39.
    Posse, Viktor
    et al.
    Chromosome Replication Laboratory, The Francis Crick Institute, London, United Kingdom; Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Diffley, John F. X.
    Chromosome Replication Laboratory, The Francis Crick Institute, London, United Kingdom.
    Eukaryotic DNA replication with purified budding yeast proteins2021In: 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.

  • 40. Pursell, Zachary F
    et al.
    Isoz, Isabelle
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kunkel, Thomas A
    Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase epsilon.2007In: Nucleic Acids Research, ISSN 1362-4962, Vol. 35, no 9, p. 3076-3086Article in journal (Refereed)
  • 41. Pursell, Zachary F
    et al.
    Isoz, Isabelle
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kunkel, Thomas A
    Yeast DNA polymerase epsilon participates in leading-strand DNA replication.2007In: Science, ISSN 1095-9203, Vol. 317, no 5834, p. 127-130Article in journal (Refereed)
  • 42.
    Rentoft, Matilda
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindell, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tran, Phong
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Anna Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Buckland, Robert
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Watt, Danielle L.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Melin, Beatrice
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 17, p. 4723-4728Article in journal (Refereed)
    Abstract [en]

    Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that severalSAMHD1mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of oneSAMHD1allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associatedSAMHD1mutations increase mutation rates in cancer cells.

  • 43.
    Rentoft, Matilda
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Svensson, Daniel
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sjödin, Andreas
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Division of CBRN Security and Defence, FOI–Swedish Defence Research Agency, SE Umeå, Sweden.
    Olason, Pall I.
    Sjöström, Olle
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Unit of research, education and development, Region Jämtland Härjedalen, SE Östersund, Sweden.
    Nylander, Carin
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjögren, Rickard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Netotea, Sergiu
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Science for Life Laboratory, Department of Biology and Biological Engineering, Chalmers University of Technology, SE Göteborg, Sweden.
    Wibom, Carl
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Cederquist, Kristina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Melin, Beatrice S.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A geographically matched control population efficiently limits the number of candidate disease-causing variants in an unbiased whole-genome analysis2019In: PLOS ONE, E-ISSN 1932-6203, Vol. 14, no 3, article id e0213350Article in journal (Refereed)
    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.

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  • 44.
    Sabouri, Nasim
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Translesion synthesis of abasic sites by yeast DNA polymerase epsilon2009In: The Journal of biological chemistry, ISSN 1083-351X, Vol. 284, no 46, p. 31555-31563Article in journal (Refereed)
    Abstract [en]

    Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase epsilon bypasses a model abasic site with comparable efficiency to Pol eta and Dpo4, two translesion polymerases. DNA polymerase epsilon also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase epsilon with 3'-5' proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.

  • 45.
    Sabouri, Nasim
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Viberg, Jörgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 17, p. 5660-5667Article in journal (Refereed)
    Abstract [en]

    The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases approximately 6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase epsilon does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19-27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions.

  • 46. Shcherbakova, Polina V
    et al.
    Pavlov, Youri I
    Chilkova, Olga
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Rogozin, Igor B
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kunkel, Thomas A
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Unique error signature of the four-subunit yeast DNA polymerase epsilon.2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 44, p. 43770-43780Article in journal (Refereed)
  • 47. Sparks, Justin L
    et al.
    Chon, Hyongi
    Cerritelli, Susana M
    Kunkel, Thomas A
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Crouch, Robert J
    Burgers, Peter M
    RNase H2-Initiated Ribonucleotide Excision Repair2012In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 47, no 6, p. 980-986Article in journal (Refereed)
    Abstract [en]

    Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. Here, we present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. RER is most efficient when the ribonucleotide is incised by RNase H2, and further excised by the flap endonuclease FEN1 with strand displacement synthesis carried out by DNA polymerase δ, the PCNA clamp, its loader RFC, and completed by DNA ligase I. We observed partial redundancy for several of the enzymes in this pathway. Exo1 substitutes for FEN1 and Pol ε for Pol δ with reasonable efficiency. However, RNase H1 fails to substitute for RNase H2 in the incision step of RER.

  • 48.
    Svensson, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rentoft, Matilda
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Dahlin, Anna M.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Lundholm, Emma
    Umeå University, Faculty of Social Sciences, Centre for Demographic and Ageing Research (CEDAR).
    Olason, Pall, I
    Sjödin, Andreas
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Division of CBRN Security and Defence, FOI–Swedish Defence Research Agency, Umeå, Sweden.
    Nylander, Carin
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Melin, Beatrice S.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A whole-genome sequenced control population in northern Sweden reveals subregional genetic differences2020In: PLOS ONE, E-ISSN 1932-6203, Vol. 15, no 9, article id e0237721Article in journal (Refereed)
    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.

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  • 49.
    ter Beek, Josy
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Parkash, Vimal
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ2019In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 11, p. 5712-5722Article in journal (Refereed)
    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.

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  • 50. Wardle, Josephine
    et al.
    Burgers, Peter M J
    Cann, Isaac K O
    Darley, Kate
    Heslop, Pauline
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lin, Li-Jung
    McGlynn, Peter
    Sanvoisin, Jonathan
    Stith, Carrie M
    Connolly, Bernard A
    Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya.2008In: Nucleic Acids Research, ISSN 1362-4962, Vol. 36, no 3, p. 705-711Article in journal (Refereed)
12 1 - 50 of 55
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