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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.
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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: 2019-01-22Bibliographically 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
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1987
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: 2018-09-24Bibliographically approved
2. Biochemical analysis of Pfh1, the essential Pif1 family helicase in Schizosaccharomyces pombe
Open this publication in new window or tab >>Biochemical analysis of Pfh1, the essential Pif1 family helicase in Schizosaccharomyces pombe
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA stores the genetic information of all living organisms, and this information needs to be copied accurately and passed on to each daughter cell when a cell divides. However, the DNA replication machinery often meets obstacles in the genome that cause fork pausing and might result in DNA damage. DNA helicases are motor proteins that unwind duplex DNA structures using the energy from ATP hydrolysis. Helicases can also assist in replication fork progression by resolving obstacles that arise at hard-to-replicate sites such as tightly DNA-bound proteins, R-loops, and DNA secondary structures like G-quadruplexes (G4s). In this thesis, we focused on Schizosaccharomyces pombe DNA helicase Pfh1, which is localized in both the nucleus and the mitochondria and belongs to the evolutionary conserved Pif1 helicases. Pfh1 is an accessory replicative helicase, and the goal in this thesis was to gain a better mechanistic understanding of the role of nuclear Pfh1 (nPfh1). Our first aim was to elucidate the role of nPfh1 at G-quadruplex (G4) DNA. Aim two was to understand the function of nPfh1’s signature motif. Aim three was to characterize the role of nPfh1 in strand annealing.

Some G-rich sequences can form a four-stranded DNA structure called G4 DNA, and the S. pombe genome contains about 450 bioinformatically predicted G4 structures. We selected two of these sequences, one located in the ribosomal DNA region and one located in the telomeric DNA region, and showed that they form inter- and intramolecular G4 structures, respectively. Next, we established a method to express and purify recombinant nPfh1 and demonstrated that nPfh1 binds to and unwinds these structures. In addition, Pfh1 bound to both the ribosomal and telomeric DNA regions in vivo, suggesting that Pfh1 can bind and unwind G4 structures in vivo. The purified nPfh1 also unwound RNA/DNA more efficiently than DNA/DNA structures, suggesting that nPfh1 has the ability to unwind R-loops in vivo. nPfh1 also showed protein displacement activity, suggesting that it can remove tightly bound proteins from DNA. All of these properties of nPfh1 suggest that it is important for fork progression and for preserving genome integrity.

Furthermore, nPfh1 stimulated strand annealing, and this activity did not require ATP hydrolysis. The strand-annealing activity was higher for complementary DNA/DNA compared to RNA/DNA substrates and did not require a DNA overhang. Furthermore, by analyzing Pfh1 truncated variants we demonstrated that the N-terminus region of Pfh1 was mainly responsible for the strand-annealing activity, however the C-terminus region also possessed some strand-annealing activity. Point mutations in the Pif1 signature motif (SM) have been shown to be associated with an increased risk of breast cancer in humans and with inviable S. pombe cells. We purified several SM variants and found that the unwinding and protein displacement activities of nPfh1 were dependent on the SM, but not the strand-annealing activity, suggesting that the SM is important for functions that require ATP hydrolysis.

In conclusion, in this thesis we identified nPfh1 as a potent G4 unwinder, and this is the only G4 unwinder identified in S. pombe to date. We also provided detailed mechanistic insights into nPfh1 and its different domains, and this has enhanced our understanding of Pfh1’s role in maintaining genome integrity.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2019. p. 38
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2011
Keywords
G4, Pfh1, Genome integrity, helicase
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-155477 (URN)978-91-7855-020-3 (ISBN)
Public defence
2019-02-15, KB:E3.01, Lilla Hörsalen, KBC-huset, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg FoundationSwedish Society for Medical Research (SSMF)Wenner-Gren FoundationsThe Kempe FoundationsSwedish Research Council
Available from: 2019-01-25 Created: 2019-01-17 Last updated: 2019-01-23Bibliographically approved

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Wallgren, MarcusMohammad, Jani B.Yan, Kok-PhenPourbozorgi-Langroudi, ParhamEbrahimi, MahsaSabouri, Nasim

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