Effect of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction: optimal conditions for the formation and reversal of the CPT trapped Topoisomerase I cleavable complex.

The effects of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction were studied at an enzyme concentration range proper for evidencing, at the same time, both DNA relaxation and DNA cleavage/religation. Some of the requirements and the optimal conditions for the formation and reversal of the CPT-trapped Topoisomerase I-DNA cleavable complex are also characterized. We conclude that: 1.

Specific regulatory role of phosphorylation of calf thymus DNA-topoisomerase I smaller forms on the relaxational activity expression. Phosphorylation role on Topo I smaller forms activity.

Calf thymus Topo I is found to be associated with three active breakdown products, resolved from intact enzyme, which do not appear to be unique to one extraction procedure. They are phosphoproteins, whose enzymatic activity can be modulated through changes in phosphorylation, and which can be phosphorylated 'in vitro', by N II protein kinase, in the same five sites as the intact enzyme. Different amounts of 32P incorporated are observed however, in the corresponding sites.

Optimum DNA relaxation reaction conditions for calf thymus DNA-topoisomerase I are determined by specific enzyme features.

Reactivity and chemical properties of calf thymus topoisomerase I have been investigated with respect to enzyme ability to relax supercoiled DNA. The relaxation rate has been analyzed at optimum and relatively high salt concentration. Catalysis is processive at optimum salt concentration and distributive at a higher one; camptothecin decreases the initial rate of reaction in both salt conditions, but more so at the higher one.

Role of calf thymus DNA-topoisomerase I phosphorylation on relaxation activity expression and on DNA-protein interaction. Role of DNA-topoisomerase I phosphorylation.

Calf thymus DNA-Topoisomerase I activity was found to be altered by changing in phosphorylation: it was completely inhibited upon dephosphorylation by alkaline phosphatase, but incubation with N II protein kinase and ATP restored the relaxation activity to a level higher than that observed prior to dephosphorylation. The calf thymus Topoisomerase I-mediated DNA cleavage, induced by camptothecin, also proved to be inhibited by dephosphorylation, which, apparently, stabilizes the initial enzyme-substrate complex. We conclude that: the native protein is partially phosphorylated, the phosphorylation involvement is essential for the activity expression and also for DNA-protein interaction, changes in the degree of phosphorylation might be involved in the regulation of DNA processing; that evokes some properties of chromatinic peptide models, which bind DNA only when phosphorylated and leads to the assumption that they represent the minimum functional substrate for N II protein kinase.

Phosphorylation sites for type N II protein kinase in DNA-topoisomerase I from calf thymus.

1. Calf thymus DNA-topoisomerase I has been isolated, in an improved preparation, nearly to SDS-PAGE homogeneity, as a single major protein (100 kDa). 2.