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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.

Pfh1, the sole member of the Pif1 helicases in Schizosaccharomyces pombe, is multifunctional and essential for maintenance of both the nuclear and mitochondrial genomes. However, we lack mechanistic insights into the functions of Pfh1 and its different motifs. This paper is specifically concerned with the importance of the Pif1 signature motif (SM), a 23 amino acids motif unique to Pif1 helicases, because a single amino acid substitution in this motif is associated with increased risk of breast cancer in humans and inviability in S. pombe. Here we show that the nuclear isoform of Pfh1 (nPfh1) unwound RNA/DNA hybrids more efficiently than DNA/DNA, suggesting that Pfh1 resolves RNA/DNA structures like R-loops in vivo. In addition, nPfh1 displaced proteins from DNA and possessed strand-annealing activity. The unwinding and protein displacement activities were dependent on the SM because nPfh1 without a large portion of this motif (nPfh1-Δ21) or with the disease/inviability-linked mutation (nPfh1-L430P) lost these properties. Unexpectedly, both nPfh1-L430P and nPfh1-Δ21 still displayed binding to G-quadruplex DNA and demonstrated strand-annealing activity. Misregulated strand annealing and binding of nPfh1-L430P without unwinding are perhaps the reasons that cells expressing this allele are inviable.

Pif1 helicases are evolutionary conserved and important for maintaining genome integrity. The S. pombe Pif1 helicase, Pfh1, unwinds DNA/DNA and RNA/DNA substrates and has both protein displacement and strand annealing activities. Here, we characterized the strand annealing properties of Pfh1. Although Pfh1 showed higher strand annealing activity on complementary oligonucleotides that produced DNA/DNA substrates, it could also anneal complementary RNA and DNA oligonucleotides. Strand annealing occurred in both the presence and absence of ATP, showing that binding of ATP does not inhibit strand annealing. Analysis of Pfh1 truncated mutants showed that the strand annealing activity of Pfh1 was primarily located on the N-terminus region, but that the C-terminus region also mediated some strand annealing activity. The N-terminus region was even more efficient than full-length Pfh1 to perform strand annealing, but in contrast to the full-length Pfh1, N-terminus Pfh1 was unable to stably bind single-stranded oligonucleotides. However, both proteins efficiently bound an intermolecular G-quadruplex DNA, showing that the N-terminus region can stably bind certain oligonucleotides, but not single-stranded oligonucleotides. These data suggest that the binding preference for single-stranded oligonucleotides is not the only important factor for Pfh1 to perform efficient strand annealing, but that other properties of the protein is also important for the annealing efficiency.