RESUMEN
Post-translation modification through the SUMO pathway is cell cycle regulated, with specific SUMO conjugates accumulating in mitotic cells. The basis for this regulation, however, and its functional significance remain poorly understood. We present evidence that in budding yeast sumoylation during mitosis may be controlled through the SUMO deconjugating enzyme Smt4/Ulp2. We isolated the polo kinase Cdc5 as an Ulp2-interacting protein, and find a C-terminal region of Ulp2 is phosphorylated during mitosis in a Cdc5-dependent manner. cdc5 mutants display reduced levels of mitotic SUMO conjugates, suggesting Cdc5 may negatively regulate Ulp2 to promote sumoylation. Previously, we found one phenotype associated with ulp2 mutants is an inability to maintain chromatid cohesion at centromere-proximal chromosomal regions. We now show this defect is rescued by inactivating Cdc5, indicating Ulp2 maintains cohesion by counter-acting Cdc5 activity. The cohesinregulator Pds5 is a likely target of this pathway, as Cdc5 overproduction forces Pds5 dissociation from chromosomes and Pds5 overproduction restores cohesion in ulp2 mutants. Overall, these observations reveal Cdc5 is a novel regulator of the SUMO pathway and suggest the outlines of a broader circuitry in which Ulp2 and Cdc5 act in a mutually antagonistic fashion to modulate maintenance and dissolution of cohesion at centromeres.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Endopeptidasas/metabolismo , Mitosis , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas de Ciclo Celular/genética , Endopeptidasas/genética , Mutación , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Telomere integrity is maintained through end-protection proteins that block nuclease degradation and prevent telomeres from being recognized as DNA breaks. Although less well understood, end protection proteins may also play a role in facilitating telomere replication. Here, we show that overproduction (OP) of the yeast telomere capping protein Stn1 makes cells highly sensitive to the replication inhibitors hydroxyurea (HU) and methyl-methane sulfonate (MMS). Unexpectedly, this sensitivity corresponds with Stn1 OP blocking most, if not all, aspects of the S phase checkpoint. The checkpoint kinase Rad53 is phosphorylated with normal timing in Stn1 OP cells, indicating Stn1 does not interfere with signaling steps involved in activating the checkpoint. Part of the role of Stn1 in telomere integrity is mediated through the Pol12 subunit of DNA polymerase alpha (Pol alpha). We show that overproduced Stn1 generally associates with chromosomes in HU treated and untreated cells, and, remarkably, Stn1 chromosome binding and OP checkpoint defects are rescued in pol12 mutants. We propose Stn1 normally promotes Pol alpha activity at telomeres but can be recruited through Pol12 to nontelomeric sites when overproduced. During replication stress, the mislocalized Stn1 may inappropriately promote Pol alpha in a manner that interferes with Rad53 effector mechanisms controlling replication fork integrity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Quinasa de Punto de Control 2 , ADN Polimerasa I/metabolismo , Hidroxiurea/farmacología , Metilmetanosulfonato/farmacología , Modelos Biológicos , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Fase S , Saccharomyces cerevisiae/metabolismo , Huso Acromático , TemperaturaRESUMEN
Recombination cloning encompasses a set of technologies that transfer gene sequences between vectors through site-specific recombination. Due in part to the instability of linear DNA in bacteria, both the initial capture and subsequent transfer of gene sequences is often performed using purified recombination enzymes. However, we find linear DNAs flanked by loxP sites recombine efficiently in bacteria expressing Cre recombinase and the lambda Gam protein, suggesting Cre/lox recombination of linear substrates can be performed in vivo. As one approach towards exploiting this capability, we describe a method for constructing large (>1 x 10(6) recombinants) libraries of gene mutations in a format compatible with recombination cloning. In this method, gene sequences are cloned into recombination entry plasmids and whole-plasmid PCR is used to produce mutagenized plasmid amplicons flanked by loxP. The PCR products are converted back into circular plasmids by transforming Cre/Gam-expressing bacteria, after which the mutant libraries are transferred to expression vectors and screened for phenotypes of interest. We further show that linear DNA fragments flanked by loxP repeats can be efficiently recombined into loxP-containing vectors through this same one-step transformation procedure. Thus, the approach reported here could be adapted as general cloning method.