Anti-Rad21 Antikörper (A313)

Cohesin is a multiprotein complex essential for sister-chromatid cohesion. It plays a pivotal role in proper chromosome segregation and DNA damage repair. The mitotic behavior of cohesin is controlled through its phosphorylation, which possibly induces the dissociation of cohesin from chromosomes and enhances its susceptibility to separase. Here, we report using mass spectrometry and anti-phospho antibodies that the central domain of Rad21, the separase-target subunit of Schizosaccharomyces pombe cohesin, is regulated by various kinase-induced phosphorylation at nine residues, indicating the multiple roles for S. pombe cohesin. In vegetative and non-dividing G(0) cells, Rad21 is phosphorylated by unknown S/TP-consensus kinases, in mitotic and non-mitotic cells by polo/Plo1 and CDK, and in DNA-damaged cells by Rad3/ATR. While mitotic phosphorylation is implicated in the dissociation of Rad21 and its cleavage by separase in anaphase, the Rad3/ATR-dependent damage-induced phosphorylation occurs intensively at the time of repair completion, and only in post-replicative cells. This damage-induced Rad21 phosphorylation is involved in the recovery process of cells from checkpoint arrest, and needed for the removal of cohesin by separase after the completion of damage repair. These complex phospho-regulations of Rad21 indicate the functional significance of cohesin in cell adaptation to a variety of cellular conditions.

Securin-separase complex is required for sister chromatid separation. Securin degrades in an APC/cyclosome dependent manner. Separase is activated on the destruction of securin and cleaves cohesin. Fission yeast securin/Cut2 required for proper separase localization has the motifs for destruction and separase-binding at the N- and C-termini, respectively. We report here the third essential domain, which becomes toxic when the 76-amino acid fragment (81-156) in the middle is overproduced. The fragment inhibits separase, while separase is recruited normally and securin is destroyed. It may interfere with separase activation after destruction of securin. If the 127DIE129 stretch is substituted for AIA, the fragment toxicity and the full-length function are abolished. Interestingly, Cut2 is cleaved in a separase dependent manner if the cleavage consensus is introduced following the DIE sequence. This finding is consistent with the proposed model that the DIE region may mimic the cleavage site of separase and inhibit the activation of separase. Evidence for physical interaction between the fragment and separase is provided. A temperature sensitive mutation cut1-K73 isolated by its specific resistance to the fragment toxicity resides in the superhelical region of separase, suggesting that the catalytic site and the helical region in separase may cooperate for activation.

Sister chromatids are held together by cohesins. At anaphase, separase is activated by degradation of its inhibitory partner, securin. Separase then cleaves cohesins, thus allowing sister chromatid separation. Fission yeast securin (Cut2) has destruction boxes and a separase (Cut1) interaction site in the amino and carboxyl terminus, respectively. Here we show that securin is essential for separase stability and also for proper repair of DNA damaged by ultraviolet, X-ray and gamma-ray irradiation. The cut2(EA2) mutant is defective in the repair of ultraviolet damage lesions, although the DNA damage checkpoint is activated normally. In double mutant analysis of ultraviolet sensitivity, checkpoint kinase chk1 (ref. 9) and excision repair rad13 (ref. 10) mutants were additive with cut2(EA2), whereas recombination repair rhp51 (ref. 11) and cohesin subunit rad21 (ref. 12) mutants were not. Cohesin was hyper-modified on ultraviolet irradiation in a Rad3 kinase-dependent way. Experiments using either mutant cohesin that cannot be cleaved by separase or a protease-dead separase provide evidence that this DNA repair function of securin-separase acts through the cleavage of cohesin. We propose that the securin-separase complex might aid DNA repair by removing local cohesin in interphase cells.