RESUMO
The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Quinases Ciclina-Dependentes/genética , Dano ao DNA , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of Caenorhabditis elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface.
