RESUMO
The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.
Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/genética , Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , DNA Fúngico/metabolismo , Alelos , Sequência de Aminoácidos , Afidicolina/toxicidade , Candida albicans/efeitos dos fármacos , Candida albicans/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Proteínas Fúngicas/química , Genes Fúngicos , Hidroxiureia/toxicidade , Metanossulfonato de Metila/toxicidade , Dados de Sequência Molecular , Mutação/efeitos dos fármacos , Mutação/efeitos da radiação , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos da radiação , Ribonucleotídeo Redutases/deficiência , Raios UltravioletaRESUMO
Both G1 and mitotic cyclins have been implicated in regulating Candida albicans filamentous growth. We have investigated the functions of Grr1 whose orthologue in Saccharomyces cerevisiae is known to mediate ubiquitin-dependent degradation of the G1 cyclins Cln1 and Cln2. Here, we report that deleting C. albicans GRR1 causes significant stabilization of two G1 cyclins Ccn1 and Cln3 and pseudohyphal growth. grr1Delta cells are highly heterogeneous in length and many of them fail to separate after cytokinesis. Interestingly, some isolated rod-like G1 cells of similar sizes are present in the grr1Delta culture. Time-lapse microscopy revealed that the rod-shaped G1 cells first grew exclusively in width before budding and then the bud grew exclusively by apical extension until after cytokinesis, yielding rod-like daughter cells. Consistently, actin patches persistently localize to the bud tip until around the time of cytokinesis. Despite the pseudohyphal phenotype, grr1Delta cells respond normally to hyphal induction. Hyperphosphorylated Cln3 isoforms accumulate in grr1Delta cells, indicating that Grr1 selectively mediates their degradation in wild-type cells. grr1Delta pseudohyphal growth requires neither Hgc1 nor Swel, two important regulators of cell morphogenesis. Furthermore, the cellular level of Hof1, a protein having a role in cytokinesis, is also significantly increased in grr1Delta cells.