G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structuresin vitroand are unwound by the Pfh1 DNA helicase

Abstract

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.

Regulation of DNA Replication through Natural Impediments in the Eukaryotic Genome

“…In the absence of Pfh1, replication forks pause at G-quadruplexes, leading to DNA damage and genome instability [28]. A recent paper shows that, both telomeric and rDNA sequences from S. pombe , can form G-quadruplexes in vitro and that Pfh1 is able to unwind these structures [179]. Interestingly, a study suggested that G-quadruplexes not only pose replicative obstacles but also function as regulatory elements that aid in lagging-strand synthesis [180], and emerging evidence suggest the role as cis -acting regulatory elements of G-quadruplexes in DNA replication as well as in transcription, translation, and telomere maintenance [181].…”

The functions of the multi-tasking Pfh1Pif1 helicase

“…In vitro, nuclear Pfh1 binds to a telomeric DNA substrate consisting of GGGTTACA telomeric repeats (Wallgren et al 2016). Because pfh1 + is an essential gene (Tanaka et al 2002; Zhou et al 2002), spore clones from pfh1Δ strains divide only 1–3 times, and these strains show stable but shorter telomeres than wild-type cells (Zhou et al 2002).…”

Identification of Compounds that Selectively Stabilize Specific G‐Quadruplex Structures by Using a Thioflavin T‐Displacement Assay as a Tool

“…These sequences were selected from the S. pombe genome and are predicted to form G4 structures in vivo. [10] The sequence of the cdc13 + -promoter DNA is an evolutionarily conserved G4 motif and is found in all four avail-able fission yeast genomes. [10] However,t he sequences of the previously studied oligonucleotides started with at en-nucleotide poly-A tail, in contrast to the oligonucleotides studied herein.…”

“…[7] The positions of G4 motifs are not randomly placed in the genome,b ut are enriched at specific regions.F or instance, the G4 motif-enrichedf eatures in S. pombe include ribosomal DNA (rDNA), promoters, telomeres, 5' and 3' untranslatedr egions( UTRs), nucleosome-depleted regions, [8] and some origins of replication . [10] Theses tudies, conducted in vitro, demonstrated that the rDNA G4 motif adopts an intermolecular G4 structure and that the telomeric G4 motif adopts an intramolecular G4 structure. [10] Theses tudies, conducted in vitro, demonstrated that the rDNA G4 motif adopts an intermolecular G4 structure and that the telomeric G4 motif adopts an intramolecular G4 structure.…”

“…[9] One of the rDNA G4 motifs, which is located in the nontranscribed region of the rDNA,a nd at elomeric G4 DNA have been well studied in vitro. [10] Small molecules that stabilize G4 structures are used as tools for studying the biological functions of G4 structures. [10] Small molecules that stabilize G4 structures are used as tools for studying the biological functions of G4 structures.…”