RESUMO
In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.
Assuntos
Reparo do DNA/genética , Endonucleases/genética , Fosforilação/genética , Proteínas de Saccharomyces cerevisiae/genética , Autofagia/genética , Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Serina-Treonina Quinases/genética , Proteólise , Saccharomyces cerevisiae/genéticaRESUMO
The repair of DNA double-strand breaks (DSBs) by homologous recombination is essential for genomic stability. The first step in this process is resection of 5' strands to generate 3' single-stranded DNA intermediates. Efficient resection in budding yeast requires the Mre11-Rad50-Xrs2 (MRX) complex and the Sae2 protein, although the role of MRX has been unclear because Mre11 paradoxically has 3'â5' exonuclease activity in vitro. Here we reconstitute resection with purified MRX, Sae2 and Exo1 proteins and show that degradation of the 5' strand is catalyzed by Exo1 yet completely dependent on MRX and Sae2 when Exo1 levels are limiting. This stimulation is mainly caused by cooperative binding of DNA substrates by Exo1, MRX and Sae2. This work establishes the direct role of MRX and Sae2 in promoting the resection of 5' strands in DNA DSB repair.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/fisiologia , Endonucleases/fisiologia , Exodesoxirribonucleases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/química , Endonucleases/química , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
It has been reported that in vitro measurement of food-specific IgE can be used to accurately predict food allergy and reduce the risk associated with double-blinded placebo-controlled food challenges (DBPCFC). Our objective was to assess the performance characteristics of the Hycor Turbo-MP quantitative radioimmunoassay for food-specific IgE and to determine this method's comparability to another assay, the Pharmacia ImmunoCAP fluorescence enzyme immunoassay (FEIA). The dynamic range of the Turbo-MP assay is 0.05 to 100 IU/ml, compared to 0.35 to 100 IU/ml for the FEIA. Performance characteristics of the Turbo-MP assay (ie, reproducibility of the calibration curve, within-run precision, total precision, parallelism, and linearity) were determined using samples from the Hycor serum bank. The precision (CV) of IgE calibrator replicates was <10%. The total precision (CV) of the Turbo-MP assay ranged from 8.8% to 18.4% for specific IgE concentrations between 0.28 to 31.4 IU/ml. Testing of serial dilutions of sera with IgE specificities for egg white, cow's milk, codfish, wheat, peanut, and soybean showed that the assay is linear over the entire dynamic range. Serial dilution data (slopes of 1.01 to 1.10) showed parallelism to serial dilutions of the IgE calibrator (slope of 0.96). The Turbo-MP and FEIA methods were both used for quantitative assays of food-specific IgE in 457 serum samples obtained from a clinical reference laboratory. Comparison of specific IgE results by the Turbo-MP and FEIA methods for 6 major food allergens exhibited a slope of 0.99 (0.92 to 1.03) with a correlation coefficient of 0.81.
