THE REGULATION OF DNA TOPOISOMERASE-II BY CASEIN KINASE-II

DNA topoisomerase II is an essential nuclear enzyme required for the p roper condensation and segregation of chromosomes during mitotic and m eiotic cell division. The enzyme exists in the cell as a phosphoprotei n, and it is most highly phosphorylated in G2 and M-phases of the cell cycle. We have shown that topoisomerase II is the target of casein ki nase II (CKII) in yeast by comparison of in vivo and in vitro phosphot ryptic peptide maps. Limited proteolysis and probing with domain speci fic antibodies show that with the exception of a weakly modified resid ue between aa 660 and aa 1250, all residues modified by CKII are in th e last 200 amino acids of yeast topoisomerase II. This C-terminal doma in is the least conserved region of the enzyme and truncation of the e nzyme shows that it is nonessential for activity in vitro. However, th e fully dephosphorylated full-size protein is nearly inactive in decat enation assays, and activity can be restored by phosphorylation by CKI I. To reconcile these observations, we propose that the C-terminal reg ion is a negative regulatory domain, counteracted by phosphorylation w ithin the domain itself. To test this hypothesis we have mutagenised 1 2 potential CKII phosphoacceptor sites in the C-terminus of topoisomer ase II and introduced the mutant genes into a yeast strain which has a temperature sensitive top2 gene. The growth of the transformed strain s is monitored at nonpermissive temperature to determine whether C-ter minal phosphorylation is important for mitotic growth. In addition, we have purified the mutant enzymes to homogeneity for in vitro assays.