3.4.22.49 C1129S inactive 709220 3.4.22.49 C2029A - 638869 3.4.22.49 C2029A no autoproteolytic cleavage, unable to cleave SCC1 638865 3.4.22.49 E1483R/R1486E autoproteolytically cleaved like wild-type 638869 3.4.22.49 E1503R/R1506E autoproteolytically cleaved like wild-type 638869 3.4.22.49 E1532R/R1535E autoproteolytically cleaved like wild-type 638869 3.4.22.49 H1531A active site point mutation prevents Scc1 from being cleaved after binding 638864 3.4.22.49 additional information analysis of the homozygous zebrafish mutant cds (cease&desist), bearing a mutation in the separase gene, reveals high levels of polyploidy and aneuploidy, spindle defects, and a mitotic exit delay in mutant embryos. Carcinogenesis studies demonstrate that cds heterozygous adults have an eightfold increase in the percentage of fish bearing epithelial tumors 680013 3.4.22.49 additional information clear evidence for a non-proteolytic function of separase is provided: by separase gene deletion in mouse oocytes it is shown that separase null-oocytes are unable to either destroy chesin along chromosome arms or segregate homologous chromosomes and these oocytes are unable to complete cell division. Microinjection of wild-type separase mRNA in separase null-oocytes restores normal homologue disjunction and polar body extrusion 682065 3.4.22.49 additional information generation of human cells with one hESP allele-encoding uncleavable protein and another allele harboring a single cleavage site, the cells grow slowly owing to cell cycle delay, in particular during G2/M transition, but not when it was expected, i.e. during anaphase 718194 3.4.22.49 additional information in cells lacking separase or expressing noncleavable Scc1, arm cohesion is not efficiently removed during nocodazole arrest 681015 3.4.22.49 additional information it is shown that separase acts as a direct inhibitor of cyclin-dependent kinase 1 on liberation from the inhibitory protein securin. Blocking separase-cyclin-dependent kinase 1 complex formation by microinjection of anti-separase antibodies prevents polar-body extrusion in vertebrate oocytes 682062 3.4.22.49 additional information RNAi of sep-1 inhibits degranulation in addition to causing extensive chromosomal segregation failures. Temperature-sensitive sep-1(e2406) allele exhibits similar inhibition of degranulation, but has minimal effects on chromosome segregation 679433 3.4.22.49 additional information the loss-of-function of Esp1 activity in yeast cells could be complemented by the tobacco etch virus, TEV, protease, which is also able to cleave Scc1 thus promoting segregation of sister chromatids 718194 3.4.22.49 R1486A mutation fails to block autocatalytical cleavage 638863 3.4.22.49 R1506A mutation at autocatalytical cleavage site, mutant enzyme is still cleaved indicating the existence of a second cleavage site 638863 3.4.22.49 R1506A/R1486A mutation fails to block autocatalytical cleavage 638863 3.4.22.49 S1121A a mouse strain is created bearing a S1121A mutation: S1121A mutation causes infertility in mice, germ cells in the mutants are depleted during development. S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death 682481 3.4.22.49 S1121A mutation causes embryogenesis failure between the 8- and 16-cell stages in mice 700165 3.4.22.49 S1121A nonphosphorylatable mutant 731697 3.4.22.49 S1126A a doxycycline-induced overexpression of S1126A mutant in HEK-293 cells leads to an increase of the G2/M cell population from 24 to 49% within 24 h compared to wild-type. Giemsa-stained prometaphase-chromosomes from S1126A expressing cells are mostly separated (61%), whereas those from wild-type or T1346E/T1363E/S1399D expressing cells are almost always paired (78 and 83%, respectively) 680926 3.4.22.49 S1126A cleaved autoproteolytically to the same degree as wild-type, shows similar SCC1 cleavage activity as wild-type 638865 3.4.22.49 S1126A specifically abolished separase hyperphosphorylation in Smad3-deficient cells 700096 3.4.22.49 S1126A the mutation prevents the conformational change of the enzyme caused by phosphorylation, thereby rendering separase resistant to precipitation in mitosis 732187 3.4.22.49 S1126A/T1346A active mutant 709220 3.4.22.49 S1138A mutant containing phosphorylation site mutation compromises Cdk1-inhibitory ability 682062 3.4.22.49 S1139A mutant containing phosphorylation site mutation compromises Cdk1-inhibitory ability 682062 3.4.22.49 T1346A/T1363A/S1399A this triple mutant binds relatively more Cdk1 than S1126A but its Cdk1 binding capacity is much decreased compared to wildtype. A doxycycline-induced overexpression of the triple mutant in HEK-293 cells leads to an increase of the G2/M cell population from 24 to 37% within 24 h compared to wild-type. Giemsa-stained prometaphase-chromosomes from triple mutant expressing cells are mostly separated (61%), whereas those from wild-type or T1346E/T1363E/S1399D expressing cells are almost always paired (78 and 83%, respectively) 680926 3.4.22.49 T1346A/T1363A/S1399D the mitotic arrest phenotype caused by the T1346A/T1363A/S1399A triple mutant is largely abrogated when just residue 1399 is changed to aspartate. Thus among the phosphorylation sites within the Cdc6-like domain serine 1399 is sufficient to support Cdk1-dependent inhibition of separase 680926 3.4.22.49 T1346E/T1363E/S1399D changing the phosphorylation sites within the Cdc6-like domain to acidic residues results in a separase that can still be inhibited by Cdk1 in vivo 680926 3.4.22.49 T1346E/T1363E/S1399S the mitotic arrest phenotype caused by the T1346A/T1363A/S1399A triple mutant is largely abrogated when just residue 1399 is changed to serine. Thus among the phosphorylation sites within the Cdc6-like domain serine 1399 is sufficient to support Cdk1-dependent inhibition of separase 680926