Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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 helicase
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 Medicine, Department of Medical Biochemistry and Biophysics.
Show others and affiliations
2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 13, p. 6213-6231Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Oxford University Press, 2016. Vol. 44, no 13, p. 6213-6231
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-124639DOI: 10.1093/nar/gkw349ISI: 000382999300021PubMedID: 27185885OAI: oai:DiVA.org:umu-124639DiVA, id: diva2:953818
Available from: 2016-08-18 Created: 2016-08-18 Last updated: 2018-06-07Bibliographically approved
In thesis
1. Insights into the roles of the essential Pfh1 DNA helicase in the nuclear and mitochondrial genomes
Open this publication in new window or tab >>Insights into the roles of the essential Pfh1 DNA helicase in the nuclear and mitochondrial genomes
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Eukaryotic cells have two sets of genomes, the nuclear and mitochondrial, and both need to be accurately maintained. Also, the rate of transcription must be precisely regulated in these genomes. However, there are many natural barriers that dysregulate these processes. The aim of this thesis was to enhance our understanding of the Schizosaccharomyces pombe, Pif1 family helicase, Pfh1, and its roles in the nuclear and mitochondrial genomes. The S. pombe genome contains 446 predicted Gquadruplex (G4) structures. By circular dichroism and Thioflavin-T assay we demonstrated that sequences from the ribosomal DNA (rDNA) and telomeres form G4 structures in vitro. The recombinant nuclear isoform of Pfh1 bound and unwound these G4 structures. Also, by chromatin immunoprecipitation combined with quantitative PCR (ChIP-qPCR), we showed that Pfh1 binds these sequences in vivo. This work provides evidence that G4 structure formation in the rDNA and telomere regions is biologically important and that unwinding of G4 structures is a conserved property of Pif1 family helicases. Using ChIP-seq we found that Pfh1 binds to natural fork barriers, such as highly transcribed genes, and nucleosome depleted regions, and that replication through these sites were dependent on Pfh1. By immunoaffinity precipitation combined with mass spectrometry, Pfh1 interacted with several replisome components, as well as DNA repair proteins, and mitochondrial proteins. Furthermore, Pfh1 moved with similar kinetics as the leading strand polymerase. These findings suggest that Pfh1 is needed at natural fork barriers to promote fork progression, and that it is not just recruited to its target sites but moves with the replisome. Based on these findings, we anticipated that lack of Pfh1 would affect expression of highly transcribed genes. By performing genome-wide transcriptome analysis of S. pombe in the absence of Pfh1, we showed that highly transcribed genes are downregulated more often than other genes. Furthermore, combining absence of Pfh1 together with Topoisomerase 1 (Top1), resulted in slower cell growth, reduced DNA synthesis rate compared to single mutants, and upregulation of genes associated with DNA repair and apoptosis. These data suggest that, cells lacking both Pfh1 and Top1 have severe problem in maintaining their genomes. By ChIP-qPCR analysis we showed that Pfh1 and Top1 directly bind to mitochondrial DNA. In addition, these cells upregulated many metabolic pathways and lost about 80% of their mtDNA. These data suggest that both Pfh1 and Top1 are required for maintenance of mtDNA. This is the first evidence showing that Top1 is present in S. pombe mitochondria. In conclusion, Pfh1 directly binds mitochondrial DNA, and natural fork barriers in the nuclear DNA, such as G4 structures. In the nucleus, Pfh1 is part of the replisome. Cells lacking Pfh1 and Top1 grow slower, rapidly lose their mitochondrial DNA, have slower nuclear DNA synthesis, and induce apoptotic pathways. Finally, this thesis emphasizes the importance of both Pfh1 and Top1 in maintaining the nuclear and mitochondrial genomes.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 35
Keywords
G4, genome integrity, Pfh1, Top1, helicase, topoisomerase, replication, transcription
National Category
Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:umu:diva-147710 (URN)978-91-7601-901-6 (ISBN)
Public defence
2018-06-08, Karl Kempe Salen, KBC huset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2018-05-18 Created: 2018-05-15 Last updated: 2018-06-09Bibliographically approved

Open Access in DiVA

fulltext(9710 kB)116 downloads
File information
File name FULLTEXT01.pdfFile size 9710 kBChecksum SHA-512
19d826e3b56d81967eeea9d55bbeef1fcf05a3972b29739f4e9be6b5b3f25779589d9b66e5a4893c94731fc95dfbe2b961c70edf0afdad5d8a85124e42fa8c1f
Type fulltextMimetype application/pdf

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Wallgren, MarcusMohammad, Jani B.Yan, Kok-PhenPourbozorgi-Langroudi, ParhamEbrahimi, MahsaSabouri, Nasim
By organisation
Department of Medical Biochemistry and Biophysics
In the same journal
Nucleic Acids Research
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 116 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 302 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf