title: Characterization of an autonomously replicating sequence in Candida guilliermondii.
abstract: Candida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, biotechnological interest, and biological control potential. During a series of preliminary experiments aiming at optimizing the electroporation procedure of C. guilliermondii cells, we observed that the efficiency of transformation of an ura5 recipient strain with the corresponding dominant marker URA5 was more than a thousand fold higher as compared with the transformation of an ura3 strain with the URA3 wild type allele. This result allowed the identification of an autonomously replicating sequence (ARS) within an A/T rich region located upstream of the URA5 open reading frame (ORF). Interestingly, linear double strand DNAs (dsDNAs) containing this putative ARS are circularized and then autonomously replicated in C. guilliermondii transformed cells. We demonstrated that the C. guilliermondii Lig4p ligase, involved in the canonical non-homologous end-joining (NHEJ) pathway, was responsible for this phenomenon since a lig4 mutant was unable to circularize and to autonomously maintain transforming dsDNAs containing the putative ARS. Finally, a functional dissection of the C. guilliermondii A/T rich region located upstream of the URA5 ORF revealed the presence of a 60 bp-length sequence essential and sufficient to confer ARS properties to shuttle plasmid and linear dsDNAs.
authors.affiliation: EA2106, Biomolécules et Biotechnologies Végétales, Faculté de Pharmacie, Université François-Rabelais de Tours, France.
authors.forename: Emilien
authors.initials: E
authors.lastname: Foureau
authors.forename: Vincent
authors.initials: V
authors.lastname: Courdavault
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Navarro Gallón
authors.forename: Sébastien
authors.initials: S
authors.lastname: Besseau
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Simkin
authors.forename: Joël
authors.initials: J
authors.lastname: Crèche
authors.forename: Lucia
authors.initials: L
authors.lastname: Atehortùa
authors.forename: Nathalie
authors.initials: N
authors.lastname: Giglioli-Guivarc'h
authors.forename: Marc
authors.initials: M
authors.lastname: Clastre
authors.forename: Nicolas
authors.initials: N
authors.lastname: Papon
mesh_terms: D002175:Candida / Q000235:genetics*; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D018274:Electroporation; D010957:Plasmids; D014170:Transformation, Genetic
chemical_list: D004270:DNA, Circular; D004271:DNA, Fungal
keywords: Autonomously replicating sequence; Candida guilliermondii; LIG4; NHEJ