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

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo Cyril Ribeyre, Judith Lopes, Jean-Baptiste Boulé, Aurele Piazza, Aurore Guédin, Virginia Zakian, Jean-Louis Mergny, Alain Nicolas To cite this version: Cyril Ribeyre, Judith Lopes, Jean-Baptiste Boulé, Aurele Piazza, Aurore Guédin, et al.. The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo. PLoS Genetics, Public Library of Science, 2009, 5 (5), pp.e1000475. 10.1371/journal.pgen.1000475. hal-02109281 HAL Id: hal-02109281 https://hal.archives-ouvertes.fr/hal-02109281 Submitted on 24 Apr 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo Cyril Ribeyre1.¤, Judith Lopes1., Jean-Baptiste Boule´ 1,2, Aure` le Piazza1, Aurore Gue´din3, Virginia A. Zakian2, Jean-Louis Mergny3, Alain Nicolas1* 1 Recombinaison et Instabilite´ Ge´ne´tique, Institut Curie Centre de Recherche, CNRS UMR3244, Universite´ Pierre et Marie Curie, Paris, France, 2 Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America, 3 Laboratoire de Biophysique, Museum National d’Histoire Naturelle USM 503, INSERM U565, CNRS UMR5153, Paris, France 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 pif1D cells. Hence, we conclude that CEB1 instability in pif1D cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences. Citation: Ribeyre C, Lopes J, Boule´ J-B, Piazza A, Gue´din A, et al. (2009) The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo. PLoS Genet 5(5): e1000475. doi:10.1371/journal.pgen.1000475 Editor: Orna Cohen-Fix, National Institute of Diabetes and Digestive and Kidney Diseases, United States of America Received January 12, 2009; Accepted April 8, 2009; Published May 8, 2009 Copyright: ß 2009 Ribeyre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by La Ligue Nationale contre le Cancer ‘‘Equipe Labellise´e LIGUE 2007’’ and in part by an E.U. FP6 ‘‘MolCancerMed’’ (LSHC-CT- 2004-502943) grant. CR was supported by a graduate student fellowship from the MNERT and the Association pour la Recherche contre le Cancer (ARC). JBB was supported by a post-doctoral fellowship from the ARC. AP was supported by a graduate student fellowship from the MNERT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Department of Molecular Biology, University of Geneva, Geneva, Switzerland . These authors contributed equally to this work. Introduction stabilize their formation, such as the beta subunit of the ciliate Oxytricha telomere binding protein complex [3,4]. At the chromosomal level, in addition to coding regions and Evidence for in vivo formation of G4 DNA has emerged in epigenetic modifications, the biological information also resides in recent years. Notably, G4 DNA has been observed by electron DNA secondary structures, but this layer remains to be further microscopy from transcribed human G-rich DNA arrays in deciphered. Biophysical and structural studies have long estab- bacteria [5] and has been detected at the end of the ciliate lished that in vitro DNA can adopt diverse structures different Oxytricha telomeres by immunochemistry [6,7]. As a complemen- from the canonical Watson-Crick conformations [1]. However, for tary approach, genome-wide bioinformatic analyses have identi- a long time, the hypothesis that these structures occur in the native fied regions that have the potential to form G4 DNA within chromosomal context, as an integral part of the functional evolutionary diverse model systems, from bacteria to human. For architecture of a chromosome, has been regarded with a certain example, in the human genome, more than 300,000 distinct sites skepticism. One example of such a non canonical DNA structure is have the potential to form G4 DNA [8,9]. These sequences are the G-quadruplex, also named G-tetraplex or G4 DNA. These highly over-represented in the promoter regions of diverse structures form in vitro in guanine-rich sequences that contain four organisms, including human [10], yeast [11] and bacteria [12]. tracts of at least three guanines separated by other bases, and are In addition, potential G4-forming sequences are found in G-rich stabilized by G-quartets that form between four DNA strands [2]. arrays such as telomeres, rDNA or G-rich micro- and minisa- Under physiological conditions, long runs of G4-forming sequenc- tellites. Hence, it has been suggested that their presence might es promote the formation of highly stable structures that can form affect transcriptional or post-transcriptional events when the G4 spontaneously in vitro and, once formed, are very resistant to forming sequence is within the transcribed region [11,13]. G4 thermal denaturation. It is also important to consider that DNA has also been proposed to participate in telomere capping, sequences that form G4-DNA slowly in vitro may be more prone DNA replication and recombination [14]. However, it remains to to fold in vivo owing to the action of proteins that promote and/or be determined how and to what extent these secondary structures PLoS Genetics | www.plosgenetics.org 1 May 2009 | Volume 5 | Issue 5 | e1000475 Pif1 Unwinds G-Quadruplex Structures Author Summary helicase is to process G4 structures. As sequences with the ability to form G4 DNA are found throughout the yeast genome, beyond Changes in the primary DNA sequence are a major source acting on intrinsically instable repeats, we propose that the of pathologies and cancers. The hereditary information processing of G4 structures by Pif1 may facilitate DNA replication, also resides in secondary DNA structures, a layer of genetic transcription and/or repair. information that remains poorly understood. Biophysical and structural studies have long established that, in vitro, Results the DNA molecule can adopt diverse structures different from the canonical Watson-Crick conformations. However, The DNA Helicase Pif1 Actively Destabilizes CEB1 during for a long time their existence in vivo has been regarded Vegetative Growth with a certain skepticism and their functional role elusive. We previously developed yeast strains to study the genetic One example is the G-quadruplex structure, which involves instability of a natural 1.8 kb allele of the human minisatellite CEB1 G-quartets that form between four DNA strands. Here, inserted in the S. cerevisiae genome (Figure 1A). This allele (called using in vitro and in vivo assays in the yeast S. cerevisiae, we reveal the unexpected role of the Pif1 helicase in CEB1-1.8) is composed of a tandem array of 42 polymorphic maintaining the stability of the human CEB1 G-rich tandem repeats of sizes varying between 36 and 43 base pairs (bp) [25] repeat array. By site-directed mutagenesis, we show that (Figure S1). In our standard assay, which measures the frequency of the genomic instability of CEB1 repeats in absence of Pif1 allele size variation after growth for seven generations at 30uC, and is directly dependent on the ability of CEB1 to form G- approximately 0.3% of wild-type (WT) cells exhibit a change in quadruplex structures. We show that Pif1 is very efficient in CEB1 size (contractions and expansions). Using this system, we vitro in processing G-quadruplex structures formed by reported that CEB1-1.8 was strongly destabilized in the absence of CEB1. We propose that Pif1 maintains CEB1 repeats by its the Rad27/FEN1 endonuclease (42% instability) [26]. ability to resolve G-quadruplex structures, thus providing Recently, it was reported that the lethality caused by inactivation circumstantial evidence of their formation in vivo. of the essential helicase/endonuclease Dna2, which

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