Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.

Ikenna Obi,Karam Chand,Erik Chorell,Vandana Singh,Jan Jamroskovic,Nasim Sabouri,Fredrik Westerlund,Matilda Rentoft

doi:10.1093/nar/gkaa820

Ikenna Obi, Karam Chand + Show 6 more

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https://doi.org/10.1093/nar/gkaa820

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Abstract

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

Highlights

Nucleic acids rich in guanine bases can fold into non-canonical secondary DNA structures termed Gquadruplexes (G4s) [1]
Wildtype S. pombe cells are resistant to multiple drugs due to efficient efflux pumps that expel toxins out of the cell [48], and S. pombe cells have not been extensively used in drug discovery
We have used a range of approaches to study the formation of G4 structures in S. pombe, a fission yeast species that diverged from the budding yeast S. cerevisiae >1 billion years ago [79]

Summary

Introduction

Nucleic acids rich in guanine bases can fold into non-canonical secondary DNA structures termed Gquadruplexes (G4s) [1]. These structures are formed when four guanine residues form a planar structure––a G-tetrad––through Hoogsteen hydrogen bonds. G4 structures have high thermodynamic stability under physiological conditions [2], and the thermostability and likelihood of formation of a G4 structure are correlated with the length and sequence of the loop region that connects the G-tetrads [3,4]. Bioinformatics analyses have revealed the enrichment of predicted G4 structures in origins of replication, gene promoters, 5 and 3 untranslated regions of mRNA, meiotic double-strand break hot spots, ribosomal DNA (rDNA) and telomeres [5,6,7,8,9,10,11]. The formation of G4 structures is important for transcriptional and translational regulation, as well as for telomere maintenance [15,16], but if they are not resolved, G4 structures can induce replication stalling and genome instability [17]

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