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Insights into the roles of the essential Pfh1 DNA helicase in the nuclear and mitochondrial genomes
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Nasim Sabouri)ORCID iD: 0000-0002-5957-627X
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 [en]
G4, genome integrity, Pfh1, Top1, helicase, topoisomerase, replication, transcription
National Category
Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Identifiers
URN: urn:nbn:se:umu:diva-147710ISBN: 978-91-7601-901-6 (print)OAI: oai:DiVA.org:umu-147710DiVA, id: diva2:1205867
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
List of papers
1. 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
Open this publication in new window or tab >>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
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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
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-124639 (URN)10.1093/nar/gkw349 (DOI)000382999300021 ()27185885 (PubMedID)
Available from: 2016-08-18 Created: 2016-08-18 Last updated: 2018-06-07Bibliographically approved
2. Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity
Open this publication in new window or tab >>Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity
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2016 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 9, article id e1006238Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Copernicus GmbH, 2016
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:umu:diva-125854 (URN)10.1371/journal.pgen.1006238 (DOI)000386069000004 ()27611590 (PubMedID)
Available from: 2016-09-20 Created: 2016-09-20 Last updated: 2018-06-07Bibliographically approved
3. Topoisomerase 1 and the Pfh1 helicase are both required for proper DNA synthesis
Open this publication in new window or tab >>Topoisomerase 1 and the Pfh1 helicase are both required for proper DNA synthesis
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(English)Manuscript (preprint) (Other academic)
National Category
Bioinformatics and Systems Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-147707 (URN)
Available from: 2018-05-15 Created: 2018-05-15 Last updated: 2018-06-09
4. Top1 and Pfh1Pif1 maintain the S. pombe mitochondrial genome
Open this publication in new window or tab >>Top1 and Pfh1Pif1 maintain the S. pombe mitochondrial genome
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:umu:diva-147708 (URN)
Available from: 2018-05-15 Created: 2018-05-15 Last updated: 2018-06-09

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