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Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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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. Vol. 12, no 9, article id e1006238
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:umu:diva-125854DOI: 10.1371/journal.pgen.1006238ISI: 000386069000004PubMedID: 27611590OAI: oai:DiVA.org:umu-125854DiVA, id: diva2:972290
Available from: 2016-09-20 Created: 2016-09-20 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)
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Available from: 2018-05-18 Created: 2018-05-15 Last updated: 2018-06-09Bibliographically approved

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