RESUMEN
The meiosis-specific HOP1 gene is important both for crossing over between homologs and for production of viable spores. hop1 diploids fail to assemble synaptonemal complex (SC), which normally provides the framework for meiotic synapsis. Immunochemical methods have shown that the 70-kDa HOP1 product is a component of the SC. To assess its molecular function, we have purified Hop1 protein to homogeneity and shown that it forms dimers and higher oligomers in solution. Consistent with the zinc-finger motif in its sequence, the purified protein contained about 1 mol equivalent of zinc whereas mutant protein lacking a conserved cysteine within this motif did not. Electrophoretic gel mobility shift assays with different forms of M13 DNA showed that Hop1 binds more readily to linear duplex DNA and negatively superhelical DNA than to nicked circular duplex DNA and even more weakly to single-stranded DNA. Linear duplex DNA binding was enhanced by the addition of Zn2+, was stronger for longer DNA fragments, and was saturable to about 55 bp/protein monomer. Competitive inhibition of this binding by added oligonucleotides suggests preferential affinity for G-rich sequences and weaker binding to poly(dA-dT). Nuclear extracts of meiotic cells caused exonucleolytic degradation of linear duplex DNA if the extracts were prepared from hop1 mutants; addition of purified Hop1 conferred protection against this degradation. These findings suggest that Hop1 acts in meiotic synapsis by binding to sites of double-strand break formation and helping to mediate their processing in the pathway to meiotic recombination.
Asunto(s)
ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Complejo Sinaptonémico , Cationes Bivalentes , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Exonucleasas/metabolismo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Expresión Génica , Magnesio , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/genética , Relación Estructura-Actividad , ZincRESUMEN
BACKGROUND: Vegetatively dividing cells of Saccharomyces cerevisiae carrying a mutation in RAD50 grow significantly more slowly in rich medium and are sensitive to DNA damage inflicted by X-ray or chemical mutagens. RAD50 function is essential for the formation and repair of meiosis-specific double-strand breaks and chromosome stability. RESULTS: We present evidence for two new phenotypes associated with the rad50delta mutant; shortened telomeres and cell senescence. Comparison of TG1-3 telomeric sequences in an isogenic pair of RAD50 and rad50delta haploid strains showed that they were considerably shortened in the latter. Although rad50delta mutation conferred cell enlargement and slow growth, cell doubling was faster but caused an increase in the frequency of cell death. Telomeres were restored to the wild-type size in hemizygous RAD50/rad50delta and rad50S/rad50delta strains; however, they showed a significant increase in rad50S/rad50S diploid with a concomitant rise in cell viability. Telomeres were stabilized in hemizygous RAD50/rad50delta and rad50S/rad50delta diploids during prolonged growth, suggesting that even a half-dosage of RAD50 is sufficient to conserve the telomere size during successive cell divisions. Furthermore, cells bearing the rad50delta mutation revealed abnormalities in nuclear segregation and, in the presence of hydroxyurea, displayed phenotypes consistent with defects in S-phase checkpoint control. CONCLUSION: This report presents evidence of the involvement of a gene relevant to recombinational repair in the maintenance of telomeres. We conclude that the phenotypes displayed by yeast rad50delta cells have intriguing similarities among the human cell lines representing DNA repair-deficient chromosome instability syndromes.
Asunto(s)
Senescencia Celular/genética , Senescencia Celular/fisiología , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Genes Fúngicos , Mutación , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Telómero/genética , Telómero/fisiología , Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , FenotipoRESUMEN
Telomeres are the termini of linear eukaryotic chromosomes consisting of tandem repeats of DNA and proteins that bind to these repeat sequences. Telomeres ensure the complete replication of chromosome ends, impart protection to ends from nucleolytic degradation, end-to-end fusion, and guide the localization of chromosomes within the nucleus. In addition, a combination of genetic, biochemical, and molecular biological approaches have implicated key roles for telomeres in diverse cellular processes such as regulation of gene expression, cell division, cell senescence, and cancer. This review focuses on recent advances in our understanding of the organization of telomeres, telomere replication, proteins that bind telomeric DNA, and the establishment of telomere length equilibrium.