RESUMO
Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have previously been characterized: it recruits telomerase to the telomere and protects chromosome ends from degradation. In a synthetic lethality screen with YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we identified a new mutation in CDC13, cdc13-4, that points toward an additional regulatory function of CDC13. Although CDC13 is an essential telomerase component in vivo, no replicative senescence can be observed in cdc13-4 cells. Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach a stable level. Thus, in cdc13-4 mutants, telomerase seems to be inhibited at normal telomere length but fully active at short telomeres. Furthermore, chromosome end structure remains protected in cdc13-4 mutants. Progressive telomere shortening to a steady-state level has also been described for mutants of the positive telomere length regulator TEL1. Strikingly, cdc13-4/tel1Delta double mutants display shorter telomeres than either single mutant after 125 generations and a significant amplification of Y' elements after 225 generations. Therefore CDC13, TEL1, and the Yku heterodimer seem to represent distinct pathways in telomere length maintenance. Whereas several CDC13 mutants have been reported to display elongated telomeres indicating that Cdc13p functions in negative telomere length control, we report a new mutation leading to shortened and eventually stable telomeres. Therefore we discuss a key role of CDC13 not only in telomerase recruitment but also in regulating telomerase access, which might be modulated by protein-protein interactions acting as inhibitors or activators of telomerase activity.
Assuntos
Ciclina B/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Telomerase/metabolismo , Telômero/metabolismo , Southern Blotting , Ciclina B/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactose/metabolismo , Marcação de Genes , Glucose/metabolismo , Mutação , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Telomerase/genéticaRESUMO
The Yku heterodimer from Saccharomyces cerevisiae, comprising Yku70p and Yku80p, is involved in the maintenance of a normal telomeric DNA end structure and is an essential component of nonhomologous end joining (NHEJ). To investigate the role of the Yku70p subunit in these two different pathways, we generated C-terminal deletions of the Yku70 protein and examined their ability to complement the phenotypes of a yku70(-) strain. Deleting only the 30 C-terminal amino acids of Yku70p abolishes Yku DNA binding activity and causes a yku(-) phenotype; telomeres are shortened, and NHEJ is impaired. Using conditions in which at least as much mutant protein as full-length protein is normally detectable in cell extracts, deleting only 25 C-terminal amino acids of Yku70p results in no measurable effect on DNA binding of the Yku protein, and the cells are fully proficient for NHEJ. Nevertheless, these cells display considerably shortened telomeres, and significant amounts of single-stranded overhangs of the telomeric guanosine-rich strands are observed. Co-overexpression of this protein with Yku80p could rescue some but not all of the telomere-related phenotypes. Therefore, the C-terminal domain in Yku70p defines at least one domain that is especially involved in telomere maintenance but not in NHEJ.
Assuntos
Antígenos Nucleares , DNA Helicases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Telômero/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Clonagem Molecular , Reparo do DNA , Autoantígeno Ku , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Deleção de Sequência , Telômero/ultraestruturaRESUMO
The HDF1 protein of Saccharomyces cerevisiae shares biochemical properties and structural homology with the 70-kDa subunit of the human autoantigen Ku. The Ku protein, a heterodimer composed of a 70-kDa subunit and an 80-kDa subunit, has been identified as the regulatory subunit of the DNA-dependent protein kinase. This enzyme has recently been shown to be involved in DNA repair and recombination processes in mammalian cells. Here we show that hdf1-disrupted S. cerevisiae strains are strongly sensitive toward the radiomimetic antibiotic bleomycin. In addition, mating-type switching and rates of spontaneous mitotic recombination are strongly reduced. This phenotype is similar to that of mammalian cells lacking components of the DNA-dependent protein kinase holoenzyme, suggesting that HDF1 participates in and exerts equivalent functions in S. cerevisiae.
