Abstract
BackgroundG-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo.ResultsWe determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites.ConclusionsIn the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0101-5) contains supplementary material, which is available to authorized users.
Highlights
G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds
Using chromatin immunoprecipitation in combination with deep sequencing (ChIP-seq), we found that Pfh1 was bound near approximately 20% of the G4 motifs in the assembled S. pombe nuclear genome and that fork slowing and DNA damage, as indicated by association with Cdc20, the leading strand DNA polymerase, and phosphorylated H2A (γ-H2A), respectively, were associated with G4 motifs in Pfh1-depleted cells
The mean P-value decreased from 0.003 in Pfh1-expressing cells to 1E-14 in Pfh1-depleted cells, and the peak P-values in Pfh1-depleted cells were consistently more significant [see Additional file 4; P approximately 0, Wilcoxon signedrank test]. These findings suggest that the probability of DNA damage at a given G4 motif is higher in Pfh1depleted versus Pfh1-expressing cells
Summary
G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. The budding yeast Pif DNA helicase, as well as several bacterial Pif family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo. DNA helicases are essential for genome stability. A point mutation in the human PIF1 (hPIF1) DNA helicase (hPIF1 L319P) is present in certain families with increased risk of breast cancer and not detected in unaffected controls [2]. This mutation changes a conserved residue within the 21-amino acid Pif signature motif that characterizes this family of DNA helicases [3,4]
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