CELL-CYCLE-DEPENDENT PHOSPHORYLATION AND ACTIVITY OF CHINESE-HAMSTER OVARY TOPOISOMERASE-II

Cell-cycle-dependent protein levels and phosphorylation of DNA topoiso merase II in relation to its catalytic and cleavage activities were st udied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. Whe n the phosphorylation of topoisomerase II in vivo was corrected for di fferences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered signif icantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the c ell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as ta-D-g lucopyranosyl)-4'-demethylepipodophyllotoxin (VP-16)-induced DNA doubl e-strand breaks (determined by neutral filter elution) increased in th e order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity , assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells , and maximal in M phase. Topoisomerase II decatenation activity was 1 .6-1.8-fold greater in S-, G2- and M-phase populations relative to asy nchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cy cle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between i mmunoreactive topoisomerase II protein levels, adjusted relative phosp horylation, catalytic activity, cleavage activity and amino acid resid ue(s) modified, suggests that the site of phosphorylation may be cell- cycle-dependent and critical in determining catalytic and cleavage act ivity.