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  • 1. Al-Behadili, Ali
    et al.
    Uhler, Jay P.
    Berglund, Anna-Karin
    Peter, Bradley
    Doimo, Mara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Reyes, Aurelio
    Wanrooij, Sjoerd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Zeviani, Massimo
    Falkenberg, Maria
    A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL2018Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 18, s. 9471-9483Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5′-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1.

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  • 2. Arenz, Stefan
    et al.
    Abdelshahid, Maha
    Sohmen, Daniel
    Payoe, Roshani
    Starosta, Agata L.
    Berninghausen, Otto
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
    Beckmann, Roland
    Wilson, Daniel N.
    The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis2016Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, nr 13, s. 6471-6481Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Under stress conditions, such as nutrient starvation, deacylated tRNAs bound within the ribosomal A-site are recognized by the stringent factor RelA, which converts ATP and GTP/GDP to (p)ppGpp. The signaling molecules (p) ppGpp globally rewire the cellular transcriptional program and general metabolism, leading to stress adaptation. Despite the additional importance of the stringent response for regulation of bacterial virulence, antibiotic resistance and persistence, structural insight into how the ribosome and deacylated-tRNA stimulate RelA-mediated (p)ppGpp has been lacking. Here, we present a cryo-EM structure of RelA in complex with the Escherichia coli 70S ribosome with an average resolution of 3.7 angstrom and local resolution of 4 to > 10 angstrom for RelA. The structure reveals that RelA adopts a unique 'open' conformation, where the C-terminal domain (CTD) is intertwined around an A/T-like tRNA within the intersubunit cavity of the ribosome and the N-terminal domain (NTD) extends into the solvent. We propose that the open conformation of RelA on the ribosome relieves the autoinhibitory effect of the CTD on the NTD, thus leading to stimulation of (p)ppGpp synthesis by RelA.

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  • 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 universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Parkash, Vimal
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    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 universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    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 levels2022Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, nr 14, s. 8023-8040Artikel i tidskrift (Refereegranskat)
    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.
    Barrasa, Juan I.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kahn, Tatyana G.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lundkvist, Moa J.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Schwartz, Yuri B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    DNA elements tether canonical Polycomb Repressive Complex 1 to human genes2023Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, nr 21, s. 11613-11633Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Development of multicellular animals requires epigenetic repression by Polycomb group proteins. The latter assemble in multi-subunit complexes, of which two kinds, Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2), act together to repress key developmental genes. How PRC1 and PRC2 recognize specific genes remains an open question. Here we report the identification of several hundreds of DNA elements that tether canonical PRC1 to human developmental genes. We use the term tether to describe a process leading to a prominent presence of canonical PRC1 at certain genomic sites, although the complex is unlikely to interact with DNA directly. Detailed analysis indicates that sequence features associated with PRC1 tethering differ from those that favour PRC2 binding. Throughout the genome, the two kinds of sequence features mix in different proportions to yield a gamut of DNA elements that range from those tethering predominantly PRC1 or PRC2 to ones capable of tethering both complexes. The emerging picture is similar to the paradigmatic targeting of Polycomb complexes by Polycomb Response Elements (PREs) of Drosophila but providing for greater plasticity. [GRAPHICS]

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  • 5.
    Blomberg, Jeanette
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Aguilar, Ximena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rautio, Linn
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 13, s. 5938-5950Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

  • 6.
    Boccaletto, Pietro
    et al.
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Stefaniak, Filip
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Ray, Angana
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Cappannini, Andrea
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Mukherjee, Sunandan
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Purta, Elżbieta
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Kurkowska, Małgorzata
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Shirvanizadeh, Niloofar
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    Destefanis, Eliana
    Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy.
    Groza, Paula
    Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Avşar, Gülben
    Department of Bioengineering, Gebze Technical University, Kocaeli, Turkey.
    Romitelli, Antonia
    Core Research Laboratory, ISPRO-Institute for Cancer Research, Prevention and Clinical Network, Firenze, Italy; Department of Medical Biotechnologies, Università di Siena.
    Pir, Pınar
    Department of Bioengineering, Gebze Technical University, Kocaeli, Turkey.
    Dassi, Erik
    Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy.
    Conticello, Silvestro G.
    Core Research Laboratory, ISPRO-Institute for Cancer Research, Prevention and Clinical Network, Firenze, Italy; Institute of Clinical Physiology, National Research Council, Pisa, Italy.
    Aguilo, Francesca
    Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bujnicki, Janusz M.
    Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.
    MODOMICS: a database of RNA modification pathways. 2021 update2022Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, nr D1, s. D231-D235Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The MODOMICS database has been, since 2006, a manually curated and centralized resource, storing and distributing comprehensive information about modified ribonucleosides. Originally, it only contained data on the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. Over the years, prompted by the accumulation of new knowledge and new types of data, it has been updated with new information and functionalities. In this new release, we have created a catalog of RNA modifications linked to human diseases, e.g., due to mutations in genes encoding modification enzymes. MODOMICS has been linked extensively to RCSB Protein Data Bank, and sequences of experimentally determined RNA structures with modified residues have been added. This expansion was accompanied by including nucleotide 5'-monophosphate residues. We redesigned the web interface and upgraded the database backend. In addition, a search engine for chemically similar modified residues has been included that can be queried by SMILES codes or by drawing chemical molecules. Finally, previously available datasets of modified residues, biosynthetic pathways, and RNA-modifying enzymes have been updated. Overall, we provide users with a new, enhanced, and restyled tool for research on RNA modification. MODOMICS is available at https://iimcb.genesilico.pl/modomics/.

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  • 7.
    Calvo, Patricia A
    et al.
    Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
    Martínez-Jiménez, María I
    Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
    Díaz, Marcos
    Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
    Stojkovic, Gorazd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kasho, Kazutoshi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain; Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
    Guerra, Susana
    Wanrooij, Sjoerd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Méndez, Juan
    Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
    Blanco, Luis
    Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
    Motif WFYY of human PrimPol is crucial to stabilize the incoming 3'-nucleotide during replication fork restart2021Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, nr 14, s. 8199-8213Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3'-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

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  • 8. Carrasco-Lopez, Cristian
    et al.
    Hernandez-Verdeja, Tamara
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Departamento de Biolog´ıa Medioambiental, Centro de Investigaciones Biologicas, CSIC, 28040 Madrid, Spain.
    Perea-Resa, Carlos
    Abia, David
    Catala, Rafael
    Salinas, Julio
    Environment-dependent regulation of spliceosome activity by the LSM2-8 complex in Arabidopsis2017Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 12, s. 7416-7431Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Spliceosome activity is tightly regulated to ensure adequate splicing in response to internal and external cues. It has been suggested that core components of the spliceosome, such as the snRNPs, would participate in the control of its activity. The experimental indications supporting this proposition, however, remain scarce, and the operating mechanisms poorly understood. Here, we present genetic and molecular evidence demonstrating that the LSM2-8 complex, the protein moiety of the U6 snRNP, regulates the spliceosome activity in Arabidopsis, and that this regulation is controlled by the environmental conditions. Our results show that the complex ensures the efficiency and accuracy of constitutive and alternative splicing of selected pre-mRNAs, depending on the conditions. Moreover, miss-splicing of most targeted pre-mRNAs leads to the generation of nonsense mediated decay signatures, indicating that the LSM2-8 complex also guarantees adequate levels of the corresponding functional transcripts. Interestingly, the selective role of the complex has relevant physiological implications since it is required for adequate plant adaptation to abiotic stresses. These findings unveil an unanticipated function for the LSM2-8 complex that represents a new layer of posttranscriptional regulation in response to external stimuli in eukaryotes.

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  • 9. Cerritelli, Susana M
    et al.
    Iranzo, Jaime
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Crouch, Robert J
    Tollervey, David
    El Hage, Aziz
    High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase2020Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, nr 8, s. 4274-4297Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

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  • 10.
    Chen, Changchun
    et al.
    Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
    Fenk, Lorenz A
    de Bono, Mario
    Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination2013Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, nr 20, artikel-id e193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

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  • 11. Chereji, Razvan V.
    et al.
    Bharatula, Vasudha
    Elfving, Nils
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Larsson, Miriam
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Morozov, Alexandre V.
    Broach, James R.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly2017Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 15, s. 8806-8821Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Sac-charomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.

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  • 12.
    Chylinski, Krzysztof
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Max F. Perutz Laboratories, University of Vienna, Austria .
    Makarova, Kira S.
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Koonin, Eugene V.
    Classification and evolution of type II CRISPR-Cas systems2014Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 10, s. 6091-6105Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA: crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in similar to 5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.

  • 13. Crona, Mikael
    et al.
    Moffatt, Connor
    Friedrich, Nancy C
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sjöberg, Britt-Marie
    Edgell, David R
    Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element.2011Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, nr 4, s. 1381-9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

  • 14.
    Deiana, Marco
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Andrés Castán, José María
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Josse, Pierre
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Kahsay, Abraha
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Sánchez, Darío Puchán
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Morice, Korentin
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Gillet, Natacha
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France.
    Ravindranath, Ranjitha
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France; Indian Institute for Science Education and Research (IISER), Tirupati, India.
    Patel, Ankit Kumar
    Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
    Sengupta, Pallabi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Obi, Ikenna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rodriguez-Marquez, Eva
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Khrouz, Lhoussain
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France.
    Dumont, Elise
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France; Institut Universitaire de France, 5 rue Descartes, France.
    Abad Galán, Laura
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France.
    Allain, Magali
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Walker, Bright
    Department of Chemistry, Kyung Hee University, Seoul, South Korea.
    Ahn, Hyun Seo
    Yonsei University, 50 Yonsei-ro ,Seodaemun-gu, Seoul, South Korea.
    Maury, Olivier
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France.
    Blanchard, Philippe
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Le Bahers, Tangui
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France; Institut Universitaire de France, 5 rue Descartes, France.
    Öhlund, Daniel
    Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
    von Hofsten, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Monnereau, Cyrille
    ENS de Lyon, CNRS, Laboratoire de Chimie UMR 5182, Université Claude Bernard Lyon 1, France.
    Cabanetos, Clément
    Univ Angers, CNRS, MOLTECH-ANJOU, France; Yonsei University, 50 Yonsei-ro ,Seodaemun-gu, Seoul, South Korea; Yonsei University, Building Blocks for FUture Electronics Laboratory (2BFUEL), Seoul, South Korea.
    Sabouri, Nasim
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression2023Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, nr 12, s. 6264-6285Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.

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  • 15.
    Del Peso-Santos, Teresa
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bernardo, Lisandro M D
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Skärfstad, Eleonore
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Holmfeldt, Linda
    Togneri, Peter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A hyper-mutant of the unusual σ70-Pr promoter bypasses synergistic ppGpp/DksA co-stimulation2011Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, nr 14, s. 5853-5865Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The activities of promoters can be temporally and conditionally regulated by mechanisms other than classical DNA-binding repressors and activators. One example is the inherently weak σ70-dependent Pr promoter that ultimately controls catabolism of phenolic compounds. The activity of Pr is up-regulated through the joint action of ppGpp and DksA that enhance the performance of RNA polymerase at this promoter. Here, we report a mutagenesis analysis that revealed substantial differences between Pr and other ppGpp/DksA co-stimulated promoters. In vitro transcription and RNA polymerase binding assays show that it is the T at the −11 position of the extremely suboptimal −10 element of Pr that underlies both poor binding of σ70-RNAP and a slow rate of open complex formation—the process that is accelerated by ppGpp and DksA. Our findings support the idea that collaborative action of ppGpp and DksA lowers the rate-limiting transition energy required for conversion between intermediates on the road to open complex formation.

  • 16.
    Del Peso-Santos, Teresa
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Landfors, Mattias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    Skärfstad, Eleonore
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Ryden, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Pr is a member of a restricted class of σ70-dependent promoters that lack a recognizable -10 element2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 22, s. 11308-11320Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Pr promoter is the first verified member of a class of bacterial σ(70)-promoters that only possess a single match to consensus within its -10 element. In its native context, the activity of this promoter determines the ability of Pseudomonas putida CF600 to degrade phenolic compounds, which provides proof-of-principle for the significance of such promoters. Lack of identity within the -10 element leads to non-detection of Pr-like promoters by current search engines, because of their bias for detection of the -10 motif. Here, we report a mutagenesis analysis of Pr that reveals strict sequence requirements for its activity that includes an essential -15 element and preservation of non-consensus bases within its -35 and -10 elements. We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads. However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440. Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.

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  • 17.
    del Peso-Santos, Teresa
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Inter-sigmulon communication through topological promoter coupling2016Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, nr 20, s. 9638-9649Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Divergent transcription from within bacterial intergenic regions frequently involves promoters dependent on alternative sigma-factors. This is the case for the non-overlapping sigma(70)- and sigma(54)-dependent promoters that control production of the substrate-responsive regulator and enzymes for (methyl) phenol catabolism. Here, using an array of in vivo and in vitro assays, we identify transcription-driven supercoiling arising from the sigma(54)-promoter as the mechanism underlying inter-promoter communication that results in stimulation of the activity of the sigma(70)-promoter. The non-overlapping 'back-to-back' configuration of a powerful sigma(54)-promoter and weak sigma(70)-promoter within this system offers a previously unknown means of inter-sigmulon communication that renders the sigma(70)-promoter subservient to signals that elicit sigma(54)-dependent transcription without it possessing a cognate binding site for the sigma(54)-RNA polymerase holoenzyme. This mode of control has the potential to be a prevalent, but hitherto unappreciated, mechanism by which bacteria adjust promoter activity to gain appropriate transcriptional control.

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  • 18.
    Doimo, Mara
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Department of Women and Children Health, University of Padova, Padova, Italy.
    Chaudhari, Namrata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Abrahamsson, Sanna
    Bioinformatics and Data Centre, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    L'Hôte, Valentin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nguyen, Tran V. H.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Berner, Andreas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ndi, Mama
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Abrahamsson, Alva
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Das, Rabindra Nath
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Aasumets, Koit
    Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland.
    Goffart, Steffi
    Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland.
    Pohjoismäki, Jaakko L. O.
    Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland.
    López, Marcela Dávila
    Bioinformatics and Data Centre, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wanrooij, Sjoerd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells2023Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, nr 14, s. 7392-7408Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

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  • 19.
    Elfving, Nils
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chereji, Razvan V.
    Bharatula, Vasudha
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Morozov, Alexandre V.
    Broach, James R.
    A dynamic interplay of nucleosome and Msn2 binding regulates kinetics of gene activation and repression following stress2014Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 9, s. 5468-5482Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The transcription factor Msn2 mediates a significant proportion of the environmental stress response, in which a common cohort of genes changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have applied genome-wide chromatin immunoprecipitation and nucleosome profiling to determine where Msn2 binds under stressful conditions and how that binding affects, and is affected by, nucleosome positioning. We concurrently determined the effect of Msn2 activity on gene expression following stress and demonstrated that Msn2 stimulates both activation and repression. We found that some genes responded to both intermittent and continuous Msn2 nuclear occupancy while others responded only to continuous occupancy. Finally, these studies document a dynamic interplay between nucleosomes and Msn2 such that nucleosomes can restrict access of Msn2 to its canonical binding sites while Msn2 can promote reposition, expulsion and recruitment of nucleosomes to alter gene expression. This interplay may allow the cell to discriminate between different types of stress signaling.

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  • 20. Farge, Géraldine
    et al.
    Holmlund, Teresa
    Khvorostova, Julia
    Rofougaran, Reza
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Hofer, Anders
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Falkenberg, Maria
    The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities.2008Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, nr 2, s. 393-403Artikel i tidskrift (Refereegranskat)
  • 21. Fasullo, Michael
    et al.
    Tsaponina, Olga
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sun, Mingzeng
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Elevated dNTP levels suppress hyper-recombination in Saccharomyces cerevisiae S-phase checkpoint mutants2010Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, nr 4, s. 1195-1203Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    MEC1, the essential yeast homolog of the human ATR/ATM genes, controls the S-phase checkpoint and prevents replication fork collapse at slow zones of DNA replication. The viability of hypomorphic mec1-21 is reduced in the rad52 mutant, defective in homologous recombination, suggesting that replication generates recombinogenic lesions. We previously observed a 6-, 10- and 30-fold higher rate of spontaneous sister chromatid exchange (SCE), heteroallelic recombination and translocations, respectively, in mec1-21 mutants compared to wild-type. Here we report that the hyper-recombination phenotype correlates with lower deoxyribonucleoside triphosphate (dNTP) levels, compared to wild-type. By introducing a dun1 mutation, thus eliminating inducible expression of ribonucleotide reductase in mec1-21, rates of spontaneous SCE increased 15-fold above wild-type. All the hyper-recombination phenotypes were reduced by SML1 deletions, which increase dNTP levels. Measurements of dNTP pools indicated that, compared to wild-type, there was a significant decrease in dNTP levels in mec1-21, dun1 and mec1-21 dun1, while the dNTP levels of mec1-21 sml1, mec1-21 dun1 sml1 and sml1 mutants were approximately 2-fold higher. Interestingly, higher dNTP levels in mec1-21 dun1 sml1 correlate with approximately 2-fold higher rate of spontaneous mutagenesis, compared to mec1-21 dun1. We suggest that higher dNTP levels in specific checkpoint mutants suppress the formation of recombinogenic lesions.

  • 22.
    Faucillion, Marie-Line
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Johansson, Anna-Mia
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Larsson, Jan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Modulation of RNA stability regulates gene expression in two opposite ways: through buffering of RNA levels upon global perturbations and by supporting adapted differential expression2022Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, nr 8, s. 4372-4388Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The steady state levels of RNAs, often referred to as expression levels, result from a well-balanced combination of RNA transcription and decay. Alterations in RNA levels will therefore result from tight regulation of transcription rates, decay rates or both. Here, we explore the role of RNA stability in achieving balanced gene expression and present genome-wide RNA stabilities in Drosophila melanogaster male and female cells as well as male cells depleted of proteins essential for dosage compensation. We identify two distinct RNA-stability mediated responses involved in regulation of gene expression. The first of these responds to acute and global changes in transcription and thus counteracts potentially harmful gene mis-expression by shifting the RNA stability in the direction opposite to the transcriptional change. The second response enhances inter-individual differential gene expression by adjusting the RNA stability in the same direction as a transcriptional change. Both mechanisms are global, act on housekeeping as well as non-housekeeping genes and were observed in both flies and mammals. Additionally, we show that, in contrast to mammals, modulation of RNA stability does not detectably contribute to dosage compensation of the sex-chromosomes in D. melanogaster.

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  • 23.
    Flodell, Sara
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Petersen, Michael
    Girard, Frederic
    Zdunek, Janusz
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kidd-Ljunggren, Karin
    Schleucher, Jürgen
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wijmenga, Sybren
    Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.2006Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, nr 16, s. 4449-4457Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

  • 24.
    Flodell, Sara
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Schleucher, Jurgen
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Cromsigt, Jenny
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Ippel, Hans
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Kidd-Ljunggren, Karin
    Wijmenga, Sybren
    The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges.2002Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, nr 21, s. 4803-4811Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Reverse transcription of hepatitis B virus (HBV) pregenomic RNA is essential for virus replication. In the first step of this process, HBV reverse transcriptase binds to the highly conserved encapsidation signal, epsilon (epsilon), situated near the 5' end of the pregenome. epsilon has been predicted to form a bulged stem-loop with the apical stem capped by a hexa- loop. After the initial binding to this apical stem- loop, the reverse transcriptase synthesizes a 4 nt primer using the bulge as a template. Here we present mutational and structural data from NMR on the apical stem-loop of epsilon. Application of new isotope-labeling techniques (13C/15N/2H-U-labeling) allowed resolution of many resonance overlaps and an extensive structural data set could be derived. The NMR data show that, instead of the predicted hexa-loop, the apical stem is capped by a stable UGU tri-loop closed by a C-G base pair, followed by a bulged out C. The apical stem contains therefore two unpaired pyrimidines (C1882 and U1889), rather than one as was predicted, spaced by 6 nt. C1882, the 3' neighbour to the G of the loop-closing C-G base pair, is completely bulged out, while U1889 is at least partially intercalated into the stem. Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, reveals a high degree of conservation of epsilon. In particular, the residues essential for this fold are either totally conserved or show rare non-disruptive mutations. These data strongly indicate that this fold is essential for recognition by the reverse transcriptase.

  • 25.
    Fonfara, Ines
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Curth, Ute
    Pingoud, Alfred
    Wende, Wolfgang
    Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 2, s. 847-860Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.

  • 26.
    Fonfara, Ines
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Le Rhun, Anaïs
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Chylinski, Krzysztof
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
    Makarova, Kira S.
    Lécrivain, Anne-Laure
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bzdrenga, Janek
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Koonin, Eugene V.
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems2014Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 4, s. 2577-2590Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA: Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

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  • 27. Frykholm, Karolin
    et al.
    Berntsson, Ronnie Per-Arne
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
    Claesson, Magnus
    de Battice, Laura
    Odegrip, Richard
    Stenmark, Pål
    Westerlund, Fredrik
    DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels2016Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, nr 15, s. 7219-7227Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and W Phi affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and W Phi Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.

  • 28.
    Ganai, Rais A.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. 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 universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Strand displacement synthesis by yeast DNA polymerase epsilon2016Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, nr 17, s. 8229-8240Artikel i tidskrift (Refereegranskat)
    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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  • 29.
    Ganai, Rais Ahmad
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bylund, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Johansson, Erik
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Switching between polymerase and exonuclease sites in DNA polymerase ε2015Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr 2, s. 932-942Artikel i tidskrift (Refereegranskat)
    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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  • 30. Garbacz, Marta A.
    et al.
    Cox, Phillip B.
    Sharma, Sushma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lujan, Scott A.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kunkel, Thomas A.
    The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity2019Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 8, s. 3986-3995Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.

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  • 31.
    Gnanasundram, Sivakumar Vadivel
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Malbert-Colas, Laurence
    Chen, Sa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Fusee, Leila
    Daskalogianni, Chrysoula
    Muller, Petr
    Salomao, Norman
    Fåhraeus, Robin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi. Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St. Louis, Paris, France; RECAMO, Masaryk Memorial Cancer Institute, Zlutykopec 7, Czech Republic; ICCVS, University of Gdańsk, Science, Gdańsk, Poland.
    MDM2's dual mRNA binding domains co-ordinate its oncogenic and tumour suppressor activities2020Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, nr 12, s. 6775-6787Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2’s binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2’s activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.

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  • 32. Gurvich, Olga L
    et al.
    Näsvall, S Joakim
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Baranov, Pavel V
    Björk, Glenn R
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Atkins, John F
    Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL2011Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, nr 8, s. 3079-3092Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5’-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNA(Pro) mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting.

  • 33.
    Hainzl, Tobias
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bonde, Mari
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. QureTech Bio, Umeå, Sweden.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Structural insights into CodY activation and DNA recognition2023Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, nr 14, s. 7631-7648Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands - branched chain amino acids and GTP - induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

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  • 34. Haronikova, Lucia
    et al.
    Olivares-Illana, Vanesa
    Wang, Lixiao
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Karakostis, Konstantinos
    Chen, Sa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Fåhraeus, Robin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi. RECAMO, Masaryk Memorial Cancer Institute, Brno, Czech Republic; 4Inserm U1162, Paris, France; ICCVS, University of Gdansk, Science, Gdansk, Poland.
    The p53 mRNA: an integral part of the cellular stress response2019Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 7, s. 3257-3271Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A large number of signalling pathways converge on p53 to induce different cellular stress responses that aim to promote cell cycle arrest and repair or, if the damage is too severe, to induce irreversible senescence or apoptosis. The differentiation of p53 activity towards specific cellular outcomes is tightly regulated via a hierarchical order of post-translational modifications and regulated protein-protein interactions. The mechanisms governing these processes provide a model for how cells optimize the genetic information for maximal diversity. The p53 mRNA also plays a role in this process and this review aims to illustrate how protein and RNA interactions throughout the p53 mRNA in response to different signalling pathways control RNA stability, translation efficiency or alternative initiation of translation. We also describe how a p53 mRNA platform shows riboswitch-like features and controls the rate of p53 synthesis, protein stability and modifications of the nascent p53 protein. A single cancer-derived synonymous mutation disrupts the folding of this platform and prevents p53 activation following DNA damage. The role of the p53 mRNA as a target for signalling pathways illustrates how mRNA sequences have co-evolved with the function of the encoded protein and sheds new light on the information hidden within mRNAs.

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  • 35. HARR, R
    et al.
    FALLMAN, P
    HAGGSTROM, M
    WAHLSTROM, L
    Gustafsson, Petter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    GENEUS, A COMPUTER-SYSTEM FOR DNA AND PROTEIN-SEQUENCE ANALYSIS CONTAINING AN INFORMATION-RETRIEVAL SYSTEM FOR THE EMBL DATA LIBRARY1986Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 14, nr 1, s. 273-284Artikel i tidskrift (Refereegranskat)
  • 36.
    Hogg, Matthew
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Johansson, Erik
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Promiscuous DNA synthesis by human DNA polymerase θ2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 6, s. 2611-2622Artikel i tidskrift (Refereegranskat)
    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.

  • 37.
    Hultdin, Magnus
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Grönlund, Elisabeth
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Norrback, Karl-Fredrik
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Eriksson-Lindström, E
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Just, T
    Roos, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Telomere analysis by fluorescence in situ hybridization and flow cytometry1998Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 26, nr 16, s. 3651-3656Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH, We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)(3) probe and DMA staining with propidium iodide, A simple and rapid protocol with results within 30 h was developed giving high reproducibility, One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCM telomere length values (P = 0.002), The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp, With the Q-FISHFCM method the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.

  • 38.
    Hübner, Barbara
    et al.
    School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, Singapore; NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore, Singapore.
    von Otter, Eric
    School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, Singapore; NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore, Singapore.
    Ahsan, Bilal
    School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, Singapore; NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore, Singapore.
    Wee, Mei Ling
    School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, Singapore; NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore, Singapore.
    Henriksson, Sara
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ludwig, Alexander
    School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, Singapore; NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore, Singapore.
    Sandin, Sara
    School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, Singapore; NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore, Singapore.
    Ultrastructure and nuclear architecture of telomeric chromatin revealed by correlative light and electron microscopy2022Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, nr 9, s. 5047-5063Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ.

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  • 39.
    Isoz, Isabelle
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Persson, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Volkov, Kirill
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Johansson, Erik
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 22, s. 11545-11553Artikel i tidskrift (Refereegranskat)
    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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  • 40.
    Johansson, Anna-Mia
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Allgardsson, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Stenberg, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Larsson, Jan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    msl2 mRNA is bound by free nuclear MSL complex in Drosophila melanogaster2011Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, nr 15, s. 6428-6439Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In Drosophila, the global increase in transcription from the male X chromosome to compensate for its monosomy is mediated by the male-specific lethal (MSL) complex consisting of five proteins and two non-coding RNAs, roX1 and roX2. After an initial sequence-dependent recognition by the MSL complex of 150-300 high affinity sites, the spread to the majority of the X-linked genes depends on local MSL-complex concentration and active transcription. We have explored whether any additional RNA species are associated with the MSL complex. No additional roX RNA species were found, but a strong association was found between a spliced and poly-adenylated msl2 RNA and the MSL complex. Based on our results, we propose a model in which a non-chromatin-associated partial or complete MSL-complex titrates newly transcribed msl2 mRNA and thus regulates the amount of available MSL complex by feedback. This represents a novel mechanism in chromatin structure regulation.

  • 41.
    Jurėnas, Dukas
    et al.
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, Bioénergies et Biotechnologie (IM2B), Aix-Marseille Université-CNRS, UMR 7255, Marseille, France.
    Payelleville, Amaury
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, Bioénergies et Biotechnologie (IM2B), Aix-Marseille Université-CNRS, UMR 7255, Marseille, France; DGIMI, Univ Montpellier, INRAE, Montpellier, France; Cellular and Molecular Microbiology, Faculte des Sciences, Universite libre de Bruxelles, Gosselies, Belgium.
    Roghanian, Mohammad
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Turnbull, Kathryn J.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Givaudan, Alain
    DGIMI, Univ Montpellier, INRAE, Montpellier, France.
    Brillard, Julien
    DGIMI, Univ Montpellier, INRAE, Montpellier, France.
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Experimental Medical Science, Lund University, Lund, Sweden; University of Tartu, Institute of Technology, Tartu, Estonia.
    Cascales, Eric
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, Bioénergies et Biotechnologie (IM2B), Aix-Marseille Université-CNRS, UMR 7255, Marseille, France.
    Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA2021Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, nr 14, s. 8384-8395Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.

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  • 42.
    Kahn, Tatyana G.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Dorafshan, Eshagh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Schultheis, Dorothea
    Zare, Aman
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Stenberg, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Division of CBRN Defense and Security, Swedish Defense Research Agency, FOI, Umea, 906 21, Sweden.
    Reim, Ingolf
    Pirrotta, Vincenzo
    Schwartz, Yuri B.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements2016Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, nr 21, s. 10132-10149Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.

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  • 43.
    Kasari, Villu
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Pochopien, Agnieszka A.
    Margus, Tonu
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Murina, Victoriia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Turnbull, Kathryn Jane
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Zhou, Yang
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nissan, Tracy
    Graf, Michael
    Novacek, Jiri
    Atkinson, Gemma C.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Johansson, Marcus J. O.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wilson, Daniel N.
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, 50411 Tartu, Estonia.
    A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling2019Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 16, s. 8807-8820Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3′-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

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  • 44.
    Kasho, Kazutoshi
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Stojkovic, Gorazd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Velázquez-Ruiz, Cristina
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Martínez-Jiménez, Maria Isabel
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Doimo, Mara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Laurent, Timothée
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Berner, Andreas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pérez-Rivera, Aldo E.
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Jenninger, Louise
    Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
    Blanco, Luis
    Centro de Biologia Molecular Severo Ochoa, Madrid, Spain.
    Wanrooij, Sjoerd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol2021Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, nr 4, s. 2179-2191Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.

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  • 45. Kenigsberg, Ephraim
    et al.
    Yehuda, Yishai
    Marjavaara, Lisette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Keszthelyi, Andrea
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Tanay, Amos
    Simon, Itamar
    The mutation spectrum in genomic late replication domains shapes mammalian GC content2016Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, nr 9, s. 4222-4232Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Genome sequence compositions and epigenetic organizations are correlated extensively across multiple length scales. Replication dynamics, in particular, is highly correlated with GC content. We combine genome-wide time of replication (ToR) data, topological domains maps and detailed functional epigenetic annotations to study the correlations between replication timing and GC content at multiple scales. We find that the decrease in genomic GC content at large scale late replicating regions can be explained by mutation bias favoring A/T nucleotide, without selection or biased gene conversion. Quantification of the free dNTP pool during the cell cycle is consistent with a mechanism involving replication-coupled mutation spectrum that favors AT nucleotides at late S-phase. We suggest that mammalian GC content composition is shaped by independent forces, globally modulating mutation bias and locally selecting on functional element. Deconvoluting these forces and analyzing them on their native scales is important for proper characterization of complex genomic correlations.

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  • 46. Koag, Myong-Chul
    et al.
    Nam, Kwangho
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lee, Seongmin
    The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase beta2014Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 17, s. 11233-11245Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase beta (pol beta), we report four crystal structures of pol beta complexed with dG.dTTP and dA.dCTP mismatches in the presence of Mg2+ or Mn2+. The Mg2+-bound ground-state structures show that the dA.dCTP-Mg2+ complex adopts an 'intermediate' protein conformation while the dG.dTTP-Mg2+ complex adopts an open protein conformation. The Mn2+-bound 'pre-chemistry-state' structures show that the dA.dCTP-Mn2+ complex is structurally very similar to the dA.dCTP-Mg2+ complex, whereas the dG.dTTP-Mn2+ complex undergoes a large-scale conformational change to adopt a Watson-Crick-like dG.dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that pol beta increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, pol beta appears to allow only a Watson-Crick-like conformation for purine.pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson-Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of pol beta.

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  • 47. Kochenova, Olga V
    et al.
    Bezalel-Buch, Rachel
    Tran, Phong
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Makarova, Alena V
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Burgers, Peter M J
    Shcherbakova, Polina V
    Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations2017Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 3, s. 1200-1218Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at 'normal S-phase' and 'damage-response' dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three 'X-dCTP' mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from 'S-phase' to 'damage-response' dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.

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  • 48.
    Kohler, Verena
    et al.
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Goessweiner-Mohr, Nikolaus
    Institute of Molecular Biosciences, University of Graz, Graz, Austria; Institute of Biophysics, Johannes Kepler University, Linz, Austria.
    Kohler, Andreas
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Fercher, Christian
    Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia.
    Probst, Ines
    Division of Infectious Diseases, University Medical Center Freiburg, Freiburg, Germany.
    Pavkov-Keller, Tea
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Hunger, Kristin
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Wolinski, Heimo
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Büttner, Sabrina
    Institute of Molecular Biosciences, University of Graz, Graz, Austria; Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
    Grohmann, Elisabeth
    Division of Infectious Diseases, University Medical Center Freiburg, Freiburg, Germany; Life Sciences and Technology, Beuth University of Applied Sciences, Berlin, Germany.
    Keller, Walter
    Institute of Molecular Biosciences, University of Graz, Graz, Austria; BioTechMed-Graz, Austria.
    TraN: A novel repressor of an Enterococcus conjugative type IV secretion system2018Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 17, s. 9201-9219Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The dissemination of multi-resistant bacteria represents an enormous burden on modern healthcare. Plasmid-borne conjugative transfer is the most prevalent mechanism, requiring a type IV secretion system that enables bacteria to spread beneficial traits, such as resistance to last-line antibiotics, among different genera. Inc18 plasmids, like the Gram-positive broad host-range plasmid pIP501, are substantially involved in propagation of vancomycin resistance from Enterococci to methicillin-resistant strains of Staphylococcus aureus. Here, we identified the small cytosolic protein TraN as a repressor of the pIP501-encoded conjugative transfer system, since deletion of traN resulted in upregulation of transfer factors, leading to highly enhanced conjugative transfer. Furthermore, we report the complex structure of TraN with DNA and define the exact sequence of its binding motif. Targeting this protein–DNA interaction might represent a novel therapeutic approach against the spreading of antibiotic resistances.

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  • 49.
    Koller, Timm O.
    et al.
    Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, Hamburg, Germany.
    Turnbull, Kathryn Jane
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark.
    Vaitkevicius, Karolis
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Crowe-Mcauliffe, Caillan
    Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, Hamburg, Germany.
    Roghanian, Mohammad
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark; Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Bulvas, Ondřej
    Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2, Prague 6, Czech Republic; Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Technicka 5, Prague 6, Czech Republic.
    Nakamoto, Jose A.
    Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Kurata, Tatsuaki
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Julius, Christina
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Atkinson, Gemma C.
    Department of Experimental Medical Science, Lund University, Lund, Sweden.
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Experimental Medical Science, Lund University, Lund, Sweden; University of Tartu, Institute of Technology, Tartu, Estonia.
    Wilson, Daniel N.
    Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, Hamburg, Germany.
    Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes2022Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, nr 19, s. 11285-11300Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

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  • 50.
    Kong, Ziqing
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Jia, Shaodong
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Anna Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Appelblad, Patrik
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi. Merck Chemicals and Life Science AB, Solna, Sweden.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Moritz, Thomas
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry2018Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 11, artikel-id e66Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

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