title: A study of the DNA damage checkpoint in Candida albicans: uncoupling of the functions of Rad53 in DNA repair, cell cycle regulation and genotoxic stress-induced polarized growth.
abstract: In response to genotoxic stress (GS), Candida albicans can undergo polarized growth and massive genome rearrangements including loss-of-heterozygosity (LOH) events. We evaluated the contribution of the CaRad53p and CaDun1p kinases of the DNA damage checkpoint (DDCP) in these processes. Characterization of C. albicans rad53ΔΔ and dun1ΔΔ mutants revealed that the two kinases were involved in the maintenance of heterozygosity. SNP-RFLP typing and whole-genome sequencing of rad53ΔΔ isolates having undergone a LOH revealed that, according to the chromosome on which LOH had occurred, these were predominantly due to break-induced replication/mitotic cross-over or chromosome loss. Loss of CaRAD53 also resulted in frequent aneuploidies. Deletion of CaDUN1 led to an increase in recombination-dependent LOH but did not trigger aneuploidies. It also increased GS sensitivity but did not impair GS-induced polarized growth contrary to CaRAD53 deletion. Characterization of CaRad53p site-directed mutants demonstrated that its kinase activity and N-terminal phosphorylation sites were crucial for its function in the resistance to GS, maintenance of heterozygosity, cell cycle regulation and polarized growth. Moreover, using phosphomimic mutants, we revealed an uncoupling of the functions of CaRad53p in these different processes, thus providing a novel understanding of how the DDCP may regulate downstream events in response to GS.
authors.affiliation: Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Département Génomes et Génétique, 25, rue du Docteur Roux, F-75015, Paris, France; INRA USC2019, 25, rue du Docteur Roux, F-75015, Paris, France; Univ. Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, rue du Docteur Roux, F-75015, Paris, France.
authors.forename: Raphaël
authors.initials: R
authors.lastname: Loll-Krippleber
authors.forename: Christophe
authors.initials: C
authors.lastname: d'Enfert
authors.forename: Adeline
authors.initials: A
authors.lastname: Feri
authors.forename: Dorothée
authors.initials: D
authors.lastname: Diogo
authors.forename: Aurélie
authors.initials: A
authors.lastname: Perin
authors.forename: Marina
authors.initials: M
authors.lastname: Marcet-Houben
authors.forename: Marie-Elisabeth
authors.initials: ME
authors.lastname: Bougnoux
authors.forename: Mélanie
authors.initials: M
authors.lastname: Legrand
mesh_terms: D000782:Aneuploidy; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D002453:Cell Cycle; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D019656:Loss of Heterozygosity; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000633:toxicity*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
chemical_list: D005656:Fungal Proteins; D009153:Mutagens; D064447:Checkpoint Kinase 2