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EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences. Changes in the primary DNA sequence are a major source of pathologies and cancers. The hereditary information also resides in secondary DNA structures, a layer of genetic information that remains poorly understood. Biophysical and structural studies have long established that, in vitro, the DNA molecule can adopt diverse structures different from the canonical Watson-Crick conformations. However, for a long time their existence in vivo has been regarded with a certain skepticism and their functional role elusive. One example is the G-quadruplex structure, which involves G-quartets that form between four DNA strands. Here, using in vitro and in vivo assays in the yeast S. cerevisiae, we reveal the unexpected role of the Pif1 helicase in maintaining the stability of the human CEB1 G-rich tandem repeat array. By site-directed mutagenesis, we show that the genomic instability of CEB1 repeats in absence of Pif1 and is directly dependent on the ability of CEB1 to form G-quadruplex structures. We show that Pif1 is very efficient in vitro in processing G-quadruplex structures formed by CEB1. We propose that Pif1 maintains CEB1 repeats by its ability to resolve G-quadruplex structures, thus providing circumstantial evidence of their formation in vivo.

Repair and Recombination: In most sexually reproducing organisms, the haploid gametes are produced by meiosis, a specialized cell-division during which recombination between the parental chromosomes ensures proper chromosome segregation. Double-strand breaks (DSB) generated in the DNA drive this recombination and are linked to recombination hotspots in the chromatin where histone H3 is methylated on lysine residue 4 (H3K4me) by the Set1C/COMPASS enzyme complex. Acquaviva et al. (p. 215 , published online 15 November) show that the Mer2/Mei4/Rec114 complex of the DSB generating machinery is linked to the Set1C/COMPASS enzyme complex through the Spp1 subunit, which contains a PHD-finger capable of binding to H3K4me. The Spp1 subunit recruits the Mer2 protein to sites where recombination will occur, probably by binding to both the H3K4me-marked chromatin and other factors.

G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC ₃ and Phen-DC ₆ , two recently released ligands of the bisquinolinium class. In vitro , both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo , both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N -methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo , interfere with their processing and elucidate their biological roles.

G‐quadruplexes (G4) are polymorphic four‐stranded structures formed by certain G‐rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or function in vivo are unknown. In S. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading‐strand replication. We now show that several other G4‐forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with the in vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved in C. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4‐forming sequences in vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to form in vivo. Synopsis In vivo formation of G‐quadruplex (G4) DNA structures formed by G‐rich DNA, which affects various biological processes, remains difficult to predict based on sequence features. A yeast minisatellite instability assay reveals features required for G4 DNA to form replication roadblocks inside cells. Some but not all sequences that form G4 DNA in vitro also trigger genomic instability in vivo High thermal stability of G4 structures is a key determinant of sequence instability in vivo G4 motifs bearing short pyrimidine loops are at the highest risk of causing genomic instability in eukaryotes