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Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2019 (English)In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 82, article id 102678Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2019. Vol. 82, article id 102678
Keywords [en]
G-quadruplex DNA, Schizosaccharomyces pombe, G4 stabilizer PhenDC3, Pif1 family helicase Pfh1, DNA replication, Quantitative PCR
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-162927DOI: 10.1016/j.dnarep.2019.102678Scopus ID: 2-s2.0-85071382717OAI: oai:DiVA.org:umu-162927DiVA, id: diva2:1347795
Funder
Knut and Alice Wallenberg Foundation, KAW2015-0189The Kempe Foundations, SMK1449 and SMK1632Swedish Society for Medical Research (SSMF)Available from: 2019-09-02 Created: 2019-09-02 Last updated: 2019-09-03Bibliographically approved

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Jamroskovic, JanObi, IkennaMovahedi, AnahitaChand, KaramChorell, ErikSabouri, Nasim
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