Abstract
G-quadruplexes (G4s) are four-stranded DNA structures formed by Hoogsteen base pairing between stacked sets of four guanines. Pif1 helicase plays critical roles in suppressing genomic instability in the yeast Saccharomyces cerevisiae by resolving G4s. However, the structural properties of G4s in S. cerevisiae and the substrate preference of Pif1 for different G4s remain unknown. Here, using CD spectroscopy and 83 G4 motifs from S. cerevisiae ranging in length from 30 to 60 nucleotides, we first show that G4 structures can be formed with a broad range of loop sizes in vitro and that a parallel conformation is favored. Using single-molecule FRET analysis, we then systematically addressed Pif1-mediated unwinding of various G4s and found that Pif1 is sensitive to G4 stability. Moreover, Pif1 preferentially unfolded antiparallel G4s rather than parallel G4s having similar stability. Furthermore, our results indicate that most G4 structures in S. cerevisiae sequences have long loops and can be efficiently unfolded by Pif1 because of their low stability. However, we also found that G4 structures with short loops can be barely unfolded. This study highlights the formidable capability of Pif1 to resolve the majority of G4s in S. cerevisiae sequences, narrows the fractions of G4s that may be challenging for genomic stability, and provides a framework for understanding the influence of different G4s on genomic stability via their processing by Pif1.
Highlights
G-quadruplexes (G4s) are four-stranded DNA structures formed by Hoogsteen base pairing between stacked sets of four guanines
83 G4 motifs were randomly selected from S. cerevisiae (Table S2) [4], and their folding properties were examined by circular dichroism (CD)
We discovered that DNA sequences from S. cerevisiae can form G4 structures with a broad range of loop sizes in vitro, and the parallel conformation is favored
Summary
G-quadruplexes (G4s) are four-stranded DNA structures formed by Hoogsteen base pairing between stacked sets of four guanines. The overall number of G4 DNA motifs was estimated to be 668 with loop threshold in the range of 5–50 nt [5] It is still unknown how many G4 motifs in those predicted sequences from S. cerevisiae genome may fold into G4 structures. We first showed that sequences from S. cerevisiae can form G4 structures with a broad range of loop sizes in vitro They may adopt different conformations, but parallel form is favored. Pif demonstrates very poor activity toward G4 structures with short loops as a result of their high stability and parallel conformation, suggesting this kind of G4 may be resolved by other helicases, or additional in vivo factors may assist Pif to remove them. The present work sheds new light on the formation and structural properties of G4s in S. cerevisiae and reveals the factors that may influence their removal by Pif
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