Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

Department of Food Chemistry and Biotechnology Faculty of Chemistry Brno University of Technology Purkyňova 118 61200 Brno Czech Republic

Gene Expression Regulation SSD IRCCS Ospedale Policlinico San Martino 16132 Genoa Italy

Institute of Biophysics of the Czech Academy of Sciences Královopolská 135 61200 Brno Czech Republic

Laboratory of Transcriptional Networks Department of Cellular Computational and Integrative Biology CIBIO University of Trento via Sommarive 9 38123 Trento Italy

Mutagenesis and Cancer Prevention UO IRCCS Ospedale Policlinico San Martino 16132 Genoa Italy

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