The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

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Abstract
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.