ACTIVATION OF THE XENOPUS CYCLIN DEGRADATION MACHINERY BY FULL-LENGTHCYCLIN-A

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase, In many cell types, cyclin A-associated kinase activi ty peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear, Previous work has suggested that while cyclin B is capable of trigger ing cyclin destruction in Xenopus cell-free systems, cyclin A-associat ed kinase is not able to support this function, Here we have expressed a full-length human cyclin A in Escherichia coli and purified the pro tein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B a nd incapable of protein synthesis, the temporal pattern of cyclin A-as sociated cdc2 kinase activity showed distinct differences that were de pendent on the concentration of cyclin A added, When cyclin A was adde d to a concentration that generated levels of cdc2 kinase activity cap able of inducing nuclear envelope breakdown, the histone H1 kinase act ivity profile was bi-phasic, consisting of an activation phase followe d by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction w as followed by nuclear reassembly and an additional round of DNA repli cation, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system, It has been suggested that t he evolutionary recruitment of cyclin A into an S phase function may h ave necessitated the loss of an original mitotic ability to activate t he cyclin destruction pathway, The results presented here indicate tha t cyclin A has not lost the ability to activate its own destruction an d that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating tha t there are not distinct cyclin A and cyclin B destruction pathways, T hus the ordered progression of the cell cycle requires the careful tit ration of cyclin A concentration in order to avoid activation of the c yclin destruction pathway before sufficient active cyclin B/cdc2 kinas e has accumulated.