Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro.
DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.
Kaiserman
H B
HB
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Ingebritsen
T S
TS
Benbow
R M
RM
D000818:Animals; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism*; D017886:Durapatite; D005260:Female; D006706:Homeostasis; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D010053:Ovary / Q000201:enzymology*; D010766:Phosphorylation; D014982:Xenopus laevis
D006882:Hydroxyapatites; D017886:Durapatite; D004264:DNA Topoisomerases, Type I