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  • 1. Asturias, Francisco J
    et al.
    Cheung, Iris K
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Chilkova, Olga
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wepplo, Daniel
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy.2006In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 13, no 1, p. 35-43Article in journal (Refereed)
  • 2. Bochman, Matthew L
    et al.
    Sabouri, Nasim
    Princeton Univ, Dept Mol Biol, Princeton, NJ 08544 USA .
    Zakian, Virginia A
    Unwinding the functions of the Pif1 family helicases2010In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 9, no 3, p. 237-249Article in journal (Refereed)
    Abstract [en]

    Helicases are ubiquitous enzymes found in all organisms that are necessary for all (or virtually all) aspects of nucleic acid metabolism. The Pif1 helicase family is a group of 5'-->3' directed, ATP-dependent, super family IB helicases found in nearly all eukaryotes. Here, we review the discovery, evolution, and what is currently known about these enzymes in Saccharomyces cerevisiae (ScPif1 and ScRrm3), Schizosaccharomyces pombe (SpPfh1), Trypanosoma brucei (TbPIF1, 2, 5, and 8), mice (mPif1), and humans (hPif1). Pif1 helicases variously affect telomeric, ribosomal, and mitochondrial DNA replication, as well as Okazaki fragment maturation, and in at least some cases affect these processes by using their helicase activity to disrupt stable nucleoprotein complexes. While the functions of these enzymes vary within and between organisms, it is evident that Pif1 family helicases are crucial for both nuclear and mitochondrial genome maintenance.

  • 3.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andrés Castán, José María
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Josse, Pierre
    Univ Angers, CNRS, MOLTECH-ANJOU, France.
    Kahsay, Abraha
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (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 University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Sengupta, Pallabi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rodriguez-Marquez, Eva
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (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 University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    von Hofsten, Jonas
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (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 University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 12, p. 6264-6285Article in journal (Refereed)
    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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  • 4.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Parallel G-quadruplex DNA structures from nuclear and mitochondrial genomes trigger emission enhancement in a nonfluorescent nano-aggregated fluorine-boron-based dye2023In: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 14, no 7, p. 1862-1869Article in journal (Refereed)
    Abstract [en]

    Molecular self-assembly is a powerful tool for the development of functional nanostructures with adaptive optical properties. However, in aqueous solution, the hydrophobic effects in the monomeric units often afford supramolecular architectures with typical side-by-side π-stacking arrangement with compromised emissive properties. Here, we report on the role of parallel DNA guanine quadruplexes (G4s) as supramolecular disaggregating-capture systems capable of coordinating a zwitterionic fluorine-boron-based dye and promoting activation of its fluorescence signal. The dye's high binding affinity for parallel G4s compared to nonparallel topologies leads to a selective disassembly of the dye's supramolecular state upon contact with parallel G4s. This results in a strong and selective disaggregation-induced emission that signals the presence of parallel G4s observable by the naked eye and inside cells. The molecular recognition strategy reported here will be useful for a multitude of affinity-based applications with potential in sensing and imaging systems.

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  • 5.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Das, Rabindra Nath
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A Site-Specific Self-Assembled Light-up Rotor Probe for Selective Recognition and Stabilization of c-MYC G-Quadruplex DNA2020In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 12, no 24, p. 12950-12957Article in journal (Refereed)
    Abstract [en]

    Direct and unambiguous evidence of the formation of G-quadruplexes (G4s) in human cells have shown their implication in several key biological events and has emphasized their role as important targets for small-molecule cancer therapeutics. Here, we report on the first example of a self-assembled multitasking molecular-rotor G4-binder able to discriminate between an extensive panel of G4 and non-G4 structures and to selectively light-up (up to 105-fold), bind (nanomolar range), and stabilize the c-MYC promoter G4 DNA. In particular, association with the c-MYC G4 triggers the disassembly of its supramolecular state (disaggregation-induced emission, DIE) and induces geometrical restrictions (motion-induced change in emission, MICE) leading to a significant enhancement of its emission yield. Moreover, this optical reporter is able to selectively stabilize the c-MYC G4 and inhibit DNA synthesis. Finally, by using confocal laser-scanning microscopy (CLSM) we show the ability of this compound to localize primarily in the subnuclear G4-rich compartments of cancer cells. This work provides a benchmark for the future design and development of a new generation of smart sequence-selective supramolecular G4-binders that combine outstanding sensing and stability properties, to be utilized in anti-cancer therapy.

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  • 6.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A Light‐up Logic Platform for Selective Recognition of Parallel G‐Quadruplex Structures via Disaggregation‐Induced Emission2020In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 59, no 2, p. 896-902Article in journal (Refereed)
    Abstract [en]

    The design of turn‐on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G‐quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition‐driven disaggregation (on‐signal) of an ultrabright coumarin‐quinazoline conjugate. The synthesized probe selectively lights‐up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label free molecular logic system. Finally, our molecule preferentially stains the G4‐rich nucleoli of cancer cells.

  • 7.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Unravelling the cellular emission fingerprint of the benchmark G-quadruplex-interactive compound Phen-DC32020In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 56, no 91, p. 14251-14254Article in journal (Refereed)
    Abstract [en]

    Phen-DC3 is among the most commonly used G-quadruplex (G4)-stabilizers in vitro and in cells. Here, we show that the G4-interactive binding interactions enable one to tune the optical properties of Phen-DC3 allowing the detection of G4 structures in cancer cells. This work opens up new directions for the use of Phen-DC3 as a selective G4 fluorescent reporter.

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  • 8.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Josse, Pierre
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Dalinot, Clément
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Osmolovskyi, Artem
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Marqués, Pablo Simón
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Castán, José María Andrés
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Abad Galán, Laura
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Allain, Magali
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Khrouz, Lhoussain
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Maury, Olivier
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Le Bahers, Tangui
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Blanchard, Philippe
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Dabos-Seignon, Sylvie
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France.
    Monnereau, Cyrille
    Univ Lyon, ENS de Lyon, CNRS UMR 5182, Université Claude Bernard Lyon 1, Lyon, France.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cabanetos, Clément
    Univ Angers, CNRS, MOLTECH-ANJOU, SFR MATRIX, Angers, France; IRL CNRS 2002, 2BFUEL, CNRS -Yonsei University, Seoul, South Korea.
    Site-selected thionated benzothioxanthene chromophores as heavy-atom-free small-molecule photosensitizers for photodynamic therapy2022In: Communications Chemistry, E-ISSN 2399-3669, Vol. 5, article id 142Article in journal (Refereed)
    Abstract [en]

    Photodynamic therapy is a clinically approved anticancer modality that employs a light-activated agent (photosensitizer) to generate cytotoxic reactive oxygen species (ROS). There is therefore a growing interest for developing innovative photosensitizing agents with enhanced phototherapeutic performances. Herein, we report on a rational design synthetic procedure that converts the ultrabright benzothioxanthene imide (BTI) dye into three heavy-atom-free thionated compounds featuring close-to-unit singlet oxygen quantum yields. In contrast to the BTI, these thionated analogs display an almost fully quenched fluorescence emission, in agreement with the formation of highly populated triplet states. Indeed, the sequential thionation on the BTI scaffold induces torsion of its skeleton reducing the singlet-triplet energy gaps and enhancing the spin-orbit coupling. These potential PSs show potent cancer-cell ablation under light irradiation while remaining non-toxic under dark condition owing to a photo-cytotoxic mechanism that we believe simultaneously involves singlet oxygen and superoxide species, which could be both characterized in vitro. Our study demonstrates that this simple site-selected thionated platform is an effective strategy to convert conventional carbonyl-containing fluorophores into phototherapeutic agents for anticancer PDT.

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  • 9.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mosser, Maëlle
    Le Bahers, Tangui
    Dumont, Elise
    Dudek, Marta
    Denis-Quanquin, Sandrine
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andraud, Chantal
    Matczyszyn, Katarzyna
    Monnereau, Cyrille
    Guy, Laure
    Light-induced in situ chemical activation of a fluorescent probe for monitoring intracellular G-quadruplex structures2021In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 13, no 32, p. 13795-13808Article in journal (Refereed)
    Abstract [en]

    Light-activated functional materials capable of remote control over duplex and G-quadruplex (G4) nucleic acids formation at the cellular level are still very rare. Herein, we report on the photoinduced macrocyclisation of a helicenoid quinoline derivative of binaphthol that selectively provides easy access to an unprecedented class of extended heteroaromatic structures with remarkable photophysical and DNA/RNA binding properties. Thus, while the native bisquinoline precursor shows no DNA binding activity, the new in situ photochemically generated probe features high association constants to DNA and RNA G4s. The latter inhibits DNA synthesis by selectively stabilizing G4 structures associated with oncogenic promoters and telomere repeat units. Finally, the light sensitive compound is capable of in cellulo photoconversion, localizes primarily in the G4-rich sites of cancer cells, competes with a well-known G4 binder and shows a clear nuclear co-localization with the quadruplex specific antibody BG4. This work provides a benchmark for the future design and development of a brand-new generation of light-activated target-selective G4-binders.

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  • 10.
    Deiana, Marco
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andréasson, Måns
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Tamilselvi, Shanmugam
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures2021In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 16, no 8, p. 1365-1376Article in journal (Refereed)
    Abstract [en]

    G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

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  • 11. Garg, Parie
    et al.
    Stith, Carrie M
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M
    Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication.2004In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 18, no 22, p. 2764-2773Article in journal (Refereed)
  • 12.
    Jamroskovic, Jan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Deiana, Marco
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Probing the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution2022In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 199, p. 81-91Article in journal (Refereed)
    Abstract [en]

    Cytosine-rich DNA can fold into four-stranded intercalated structures called i-motifs (iMs) under acidic conditions through the formation of hemi-protonated C:C+ base pairs. However, the folding and stability of iMs rely on many other factors that are not yet fully understood. Here, we combined biochemical and biophysical approaches to determine the factors influencing iM stability under a wide range of experimental conditions. By using high-resolution primer extension assays, circular dichroism, and absorption spectroscopies, we demonstrate that the stabilities of three different biologically relevant iMs are not dependent on molecular crowding agents. Instead, some of the crowding agents affected overall DNA synthesis. We also tested a range of small molecules to determine their effect on iM stabilization at physiological temperature and demonstrated that the G-quadruplex-specific molecule CX-5461 is also a promising candidate for selective iM stabilization. This work provides important insights into the requirements needed for different assays to accurately study iM stabilization, which will serve as important tools for understanding the contribution of iMs in cell regulation and their potential as therapeutic targets.

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  • 13.
    Jamroskovic, Jan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kumar, Rajendra
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Das, Rabindra Nath
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Akhunzianov, Almaz
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia.
    Deiana, Marco
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kasho, Kazutoshi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sulis Sato, Sebastian
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pourbozorgi-Langroudi, Parham
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mason, James E.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Medini, Paolo
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Öhlund, Daniel
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization2020In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, no 6, p. 2876-2888Article in journal (Refereed)
    Abstract [en]

    The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

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  • 14.
    Jamroskovic, Jan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Livendahl, Madeleine
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Eriksson, Jonas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Identification of Compounds that Selectively Stabilize Specific G-Quadruplex Structures by Using a Thioflavin T-Displacement Assay as a Tool2016In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 22, no 52, p. 18932-18943Article in journal (Refereed)
    Abstract [en]

    Small molecules are used in the G-quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT-displacement high-throughput screening assay to find novel and selective G4-binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4-structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.

  • 15.
    Jamroskovic, Jan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Movahedi, Anahita
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay2019In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 82, article id 102678Article in journal (Refereed)
    Abstract [en]

    In order to understand in which biological processes the four-stranded G-quadruplex (G4) DNA structures play a role, it is important to determine which predicted regions can actually adopt a G4 structure. Here, to identify DNA regions in Schizosaccharomyces pombe that fold into G4 structures, we first optimized a quantitative PCR (qPCR) assay using the G4 stabilizer, PhenDC3. We call this method the qPCR stop assay, and used it to screen for G4 structures in genomic DNA. The presence of G4 stabilizers inhibited DNA amplification in 14/15 unexplored genomic regions in S. pombe that encompassed predicted G4 structures, suggesting that at these sites the stabilized G4 structure formed an obstacle for the DNA polymerase. Furthermore, the formation of G4 structures was confirmed by complementary in vitro assays. In vivo, the S. pombe G4 unwinder Pif1 helicase, Pfh1, was associated with tested G4 sites, suggesting that the G4 structures also formed in vivo. Thus, we propose that the confirmed G4 structures in S. pombe form an obstacle for replication in vivo, and that the qPCR stop assay is a method that can be used to identify G4 structures. Finally, we suggest that the qPCR stop assay can also be used for identifying G4 structures in other organisms, as well as being adapted to screen for novel G4 stabilizers.

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  • 16.
    Livendahl, Madeleine
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Görlich, T.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Synthesis of phenanthridine spiropyrans and studies of their effects on G-quadruplex DNA2017In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 15, no 15, p. 3265-3275Article in journal (Refereed)
    Abstract [en]

    G-quadruplex (G4) DNA structures are involved in many important biological processes and can be linked to several human diseases. Drug-like low molecular weight compounds that target G4 structures are therefore interesting not only for their potential therapeutic properties but also for their potential use as chemical research tools. We report here on the development of methods to synthesize spiropyrans using a condensation-cyclisation reaction of quaternary salts of [small alpha]-methyl quinoline or phenanthridine with salicylaldehydes. Evaluation of the synthesized phenanthridine spiropyrans' interactions with G4 DNA was performed with a Thioflavin T displacement assay, circular dichroism, Taq DNA polymerase stop assay, and NMR. This revealed that the substitution pattern on the phenanthridine spiropyrans was very important for their ability to bind and stabilize G4 structures. Some of the synthesized low molecular weight spirocyclic compounds efficiently stabilized G4 structures without inducing structural changes by binding the first G-tetrad in the G4 structure.

  • 17.
    Livendahl, Madeleine
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ivanova, Svetlana
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Demirel, Peter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Design and Synthesis of 2,2'-Diindolylmethanes to Selectively Target Certain G-Quadruplex DNA Structures2016In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 22, no 37, p. 13004-13009Article in journal (Refereed)
    Abstract [en]

    G-quadruplex (G4) structures carry vital biological functions, and compounds that selectively target certain G4 structures have both therapeutic potential and value as research tools. Along this line, 2,2'-diindolylmethanes have been designed and synthesized in this work based on the condensation of 3,6- or 3,7-disubstituted indoles with aldehydes. The developed class of compounds efficiently stabilizes G4 structures without inducing conformational changes in such structures. Furthermore, the 2,2'-diindolylmethanes target certain G4 structures more efficiently than others and this G4 selectivity can be altered by chemical modifications of the compounds.

  • 18. McDonald, Karin R.
    et al.
    Guise, Amanda J.
    Pourbozorgi-Langroudi, Parham
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cristea, Ileana M.
    Zakian, Virginia A.
    Capra, John A.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity2016In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 9, article id e1006238Article in journal (Refereed)
    Abstract [en]

    Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.

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  • 19. McDonald, Karin R
    et al.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Webb, Christopher J
    Zakian, Virginia A
    The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening2014In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 24, p. 80-86Article in journal (Refereed)
    Abstract [en]

    Pif1 family helicases are evolutionary conserved 5'-3' DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.

  • 20. Miralles Fusté, Javier
    et al.
    Shi, Yonghong
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhu, Xuefeng
    Jemt, Elisabeth
    Persson, Orjan
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gustafsson, Claes M
    Falkenberg, Maria
    In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication2014In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, p. e1004832-Article in journal (Refereed)
    Abstract [en]

    Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.

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  • 21.
    Mohammad, Jani B.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Biochemical analysis of the strand annealing activity of the S. pombe Pif1 helicaseManuscript (preprint) (Other academic)
    Abstract [en]

    Pif1 helicases are evolutionary conserved and important for maintaining genome integrity. The S. pombe Pif1 helicase, Pfh1, unwinds DNA/DNA and RNA/DNA substrates and has both protein displacement and strand annealing activities. Here, we characterized the strand annealing properties of Pfh1. Although Pfh1 showed higher strand annealing activity on complementary oligonucleotides that produced DNA/DNA substrates, it could also anneal complementary RNA and DNA oligonucleotides. Strand annealing occurred in both the presence and absence of ATP, showing that binding of ATP does not inhibit strand annealing. Analysis of Pfh1 truncated mutants showed that the strand annealing activity of Pfh1 was primarily located on the N-terminus region, but that the C-terminus region also mediated some strand annealing activity. The N-terminus region was even more efficient than full-length Pfh1 to perform strand annealing, but in contrast to the full-length Pfh1, N-terminus Pfh1 was unable to stably bind single-stranded oligonucleotides. However, both proteins efficiently bound an intermolecular G-quadruplex DNA, showing that the N-terminus region can stably bind certain oligonucleotides, but not single-stranded oligonucleotides. These data suggest that the binding preference for single-stranded oligonucleotides is not the only important factor for Pfh1 to perform efficient strand annealing, but that other properties of the protein is also important for the annealing efficiency.

  • 22.
    Mohammad, Jani B.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wallgren, Marcus
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The Pif1 signature motif of Pfh1 is necessary for both protein displacement and helicase unwinding activities, but is dispensable for strand-annealing activity2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 16, p. 8516-8531Article in journal (Refereed)
    Abstract [en]

    Pfh1, the sole member of the Pif1 helicases in Schizosaccharomyces pombe, is multifunctional and essential for maintenance of both the nuclear and mitochondrial genomes. However, we lack mechanistic insights into the functions of Pfh1 and its different motifs. This paper is specifically concerned with the importance of the Pif1 signature motif (SM), a 23 amino acids motif unique to Pif1 helicases, because a single amino acid substitution in this motif is associated with increased risk of breast cancer in humans and inviability in S. pombe. Here we show that the nuclear isoform of Pfh1 (nPfh1) unwound RNA/DNA hybrids more efficiently than DNA/DNA, suggesting that Pfh1 resolves RNA/DNA structures like R-loops in vivo. In addition, nPfh1 displaced proteins from DNA and possessed strand-annealing activity. The unwinding and protein displacement activities were dependent on the SM because nPfh1 without a large portion of this motif (nPfh1-Δ21) or with the disease/inviability-linked mutation (nPfh1-L430P) lost these properties. Unexpectedly, both nPfh1-L430P and nPfh1-Δ21 still displayed binding to G-quadruplex DNA and demonstrated strand-annealing activity. Misregulated strand annealing and binding of nPfh1-L430P without unwinding are perhaps the reasons that cells expressing this allele are inviable.

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  • 23.
    Obi, Ikenna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rentoft, Matilda
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Singh, Vandana
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chand, Karam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Westerlund, Fredrik
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication2020In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, no 19, p. 10998-11015Article in journal (Refereed)
    Abstract [en]

    G-quadruplex (G4) structures are stable noncanonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancerassociated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

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  • 24.
    Prasad, Bagineni
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Das, Rabindra Nath
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kumar, Rajendra
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The Relation Between Position and Chemical Composition of Bis-Indole Substituents Determines Their Interactions With G-Quadruplex DNA2020In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 26, no 43, p. 9561-9572Article in journal (Refereed)
    Abstract [en]

    G‐quadruplex (G4) DNA structures are linked to fundamental biological processes and human diseases, which has triggered the development of compounds that affect these DNA structures. However, more knowledge is needed about how small molecules interact with G4 DNA structures. This study describes the development of a new class of bis‐indoles (3,3‐diindolyl‐methyl derivatives) and detailed studies of how they interact with G4 DNA using orthogonal assays, biophysical techniques, and computational studies. This revealed compounds that strongly bind and stabilize G4 DNA structures, and detailed binding interactions which e.g. show that charge variance can play a key role in G4 DNA binding. Furthermore, the structure‐activity relationships generated opened the possibilities to replace or introduce new substituents on the core structure, which is of key importance to optimize compound properties or introduce probes to further expand the possibilities of these compounds as tailored research tools to study G4 biology.

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  • 25.
    Prasad, Bagineni
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bhowmik, Sudipta
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Biophysics, Molecular Biology & Bioinformatics, University of Calcutta, Kolkata, India.
    Kumar, Rajendra
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Romell, Tajanena
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Flexible Versus Rigid G-Quadruplex DNA Ligands: Synthesis of Two Series of Bis-indole Derivatives and Comparison of Their Interactions with G-Quadruplex DNA2018In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 24, no 31, p. 7926-7938Article in journal (Refereed)
    Abstract [en]

    Small molecules that target G-quadruplex (G4) DNA structures are not only valuable to study G4 biology but also for their potential as therapeutics. This work centers around how different design features of small molecules can affect the interactions with G4 DNA structures, exemplified by the development of synthetic methods to bis-indole scaffolds. Our synthesized series of bis-indole scaffolds are structurally very similar but differ greatly in the flexibility of their core structures. The flexibility of the molecules proved to be an advantage compared to locking the compounds in the presumed bioactive G4 conformation. The flexible derivatives demonstrated similar or even improved G4 binding and stabilization in several orthogonal assays even though their entropic penalty of binding is higher. In addition, molecular dynamics simulations with the c-MYC G4 structure showed that the flexible compounds adapt better to the surrounding. This was reflected by an increased number of both stacking and polar interactions with both the residues in the G4 DNA structure and the DNA residues just upstream of the G4 structure.

  • 26.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The functions of the multi‑tasking Pfh1Pif1 helicase2017In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 63, no 4, p. 621-626Article, review/survey (Refereed)
    Abstract [en]

    Approximately, 1% of the genes in eukaryotic genomes encode for helicases, which make the number of helicases expressed in the cell considerably high. Helicases are motor proteins that participate in many central aspects of the nuclear and mitochondrial genomes, and based on their helicase motif conservation, they are divided into different helicase families. The Pif1 family of helicases is an evolutionarily conserved helicase family that is associated with familial breast cancer in humans. The Schizosaccharomyces pombe Pfh1 helicase belongs to the Pif1 helicase family and is a multi-tasking helicase that is important for replication fork progression through natural fork barriers, for G-quadruplex unwinding, and for Okazaki fragment maturation, and these activities are potentially shared by the human Pif1 helicase. This review discusses the known functions of the Pfh1 helicase, the study of which has led to a better understanding of nucleic acid metabolism in eukaryotes.

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  • 27.
    Sabouri, Nasim
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Capra, John A
    Zakian, Virginia A
    The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage2014In: BMC Biology, E-ISSN 1741-7007, Vol. 12, no 1, article id 101Article in journal (Refereed)
    Abstract [en]

    Background: G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo.

    Results: We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites.

    Conclusions: In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.

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  • 28.
    Sabouri, Nasim
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Translesion synthesis of abasic sites by yeast DNA polymerase epsilon2009In: The Journal of biological chemistry, ISSN 1083-351X, Vol. 284, no 46, p. 31555-31563Article in journal (Refereed)
    Abstract [en]

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

  • 29.
    Sabouri, Nasim
    et al.
    Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
    McDonald, Karin R
    Webb, Christopher J
    Cristea, Ileana M
    Zakian, Virginia A
    DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase2012In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 26, no 6, p. 581-593Article in journal (Refereed)
    Abstract [en]

    Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

  • 30.
    Sabouri, Nasim
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Viberg, Jörgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kumar, Dinesh
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 17, p. 5660-5667Article in journal (Refereed)
    Abstract [en]

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

  • 31.
    Sengupta, Pallabi
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jamroskovic, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A beginner's handbook to identify and characterize i-motif DNA2023In: Methods in enzymology / [ed] Hans Renata, Elsevier, 2023Chapter in book (Refereed)
    Abstract [en]

    Genomic DNA exhibits an innate ability to manifest diverse sequence-dependent secondary structures, serving crucial functions in gene regulation and cellular equilibrium. While extensive research has confirmed the formation of G-quadruplex structures by guanine-rich sequences in vitro and in cells, recent investigations have turned the quadruplex community's attention to the cytosine (C)-rich complementary strands that can adopt unique tetra-stranded conformation, termed as intercalated motif or i-motif. I-motifs are stabilized by hemi-protonated C:CH+ base pairs under acidic conditions. Initially, the in vivo occurrence of i-motifs was underestimated because their formation is favored at non-physiological pH. However, groundbreaking research utilizing the structure-specific iMab antibody and high-throughput sequencing have recently detected their conserved dispersion throughout the genome, challenging previous assumptions. Given the evolving nature of this research field, it becomes imperative to conduct independent in vitro experiments aimed at identifying potential i-motif formation in C-rich sequences and consolidating the findings to address the properties of i-motifs. This chapter serves as an introductory guide for the swift identification of novel i-motifs, where we present an experimental framework for investigating and characterizing i-motif sequences in vitro. In this chapter, we selected a synthetic oligonucleotide (C7T3) sequence and outlined appropriate methodologies for annealing the i-motif structure into suitable buffers. Then, we validated its formation by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopy. Finally, we provided a thorough account of the step-by-step procedures to investigate the effect of i-motif formation on the stalling or retardation of DNA replication using high resolution primer extension assays.

  • 32.
    Wallgren, Marcus
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mohammad, Jani B.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yan, Kok-Phen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pourbozorgi-Langroudi, Parham
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ebrahimi, Mahsa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 13, p. 6213-6231Article in journal (Refereed)
    Abstract [en]

    Certain guanine-rich sequences have an inherent propensity to form G-quadruplex (G4) structures. G4 structures are e.g. involved in telomere protection and gene regulation. However, they also constitute obstacles during replication if they remain unresolved. To overcome these threats to genome integrity, organisms harbor specialized G4 unwinding helicases. In Schizosaccharomyces pombe, one such candidate helicase is Pfh1, an evolutionarily conserved Pif1 homolog. Here, we addressed whether putative G4 sequences in S. pombe can adopt G4 structures and, if so, whether Pfh1 can resolve them. We tested two G4 sequences, derived from S. pombe ribosomal and telomeric DNA regions, and demonstrated that they form inter- and intramolecular G4 structures, respectively. Also, Pfh1 was enriched in vivo at the ribosomal G4 DNA and telomeric sites. The nuclear isoform of Pfh1 (nPfh1) unwound both types of structure, and although the G4-stabilizing compound Phen-DC3 significantly enhanced their stability, nPfh1 still resolved them efficiently. However, stable G4 structures significantly inhibited adenosine triphosphate hydrolysis by nPfh1. Because ribosomal and telomeric DNA contain putative G4 regions conserved from yeasts to humans, our studies support the important role of G4 structure formation in these regions and provide further evidence for a conserved role for Pif1 helicases in resolving G4 structures.

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  • 33.
    Yan, Kok-Phen
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization2021In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, no 14, p. 8339-8354Article in journal (Refereed)
    Abstract [en]

    The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization.

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