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

Abstract

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. We conclude that: 1. calf thymus topoisomerase I requires, for its maximum reactivity, specific and characteristic reaction conditions; 2. salt concentration affects DNA processing, indeed influencing the initial rate of DNA relaxation and directly reflecting the salt-dependence for the enzyme-duplex DNA binding; 3. topoisomerase I, from various sources, maybe individually responds to alteration of assay parameters such as pH, Mg++ and NaCl concentrations, indicating that individual criteria could be responsible for the catalytic activity optimum.

Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I

“…Specifically dephosphorylation reportedly decreased or abolished topo I activity (13)(14)(15), and subsequent treatment with CKII or PKC␣ stimulated activity (14,15). On the other hand, recombinant topo I, which has no detectable phosphorylation, was observed to be enzymatically active (44 -46), casting doubt on the prior claim that phosphorylation is required for activity.…”

“…Treatment with calf intestine alkaline phosphatase decreases topo I enzymatic activity in vitro (13)(14)(15). Conversely subsequent treatment with PKC or CKII, two kinases that copurify with topo I and phosphorylate it in vitro (16 -18), stimulates topo I activity 2-3-fold (14,15,19) and enhances the ability of CPT to trap covalent topo I-DNA cleavage complexes (20), suggesting that phosphorylation by these kinases might make topo I more sensitive to CPT.…”

“…Treatment with calf intestine alkaline phosphatase decreases topo I enzymatic activity in vitro (13-15). Conversely subsequent treatment with PKC or CKII, two kinases that copurify with topo I and phosphorylate it in vitro (16 -18), stimulates topo I activity 2-3-fold (14,15,19) and enhances the ability of CPT to trap covalent topo I-DNA cleavage complexes (20), suggesting that phosphorylation by these kinases might make topo I more sensitive to CPT.Despite its potential importance, many aspects of topo I phosphorylation remain poorly understood. The number of phosphorylation sites, for example, remains unclear because the number of phosphopeptides detected after metabolic labeling and immunoprecipitation has varied from one (16) to as many as five (21) or six (17).…”

Molecular Basis of the Supercoil Deficit Induced by the Mini-F Plasmid Partition Complex

“…Although near maximum relaxation of most substrate molecules throughout the SopB concentration range (Fig. 3A) is not the action expected of a directly inhibited enzyme, Topo I is reported to be processive in the buffer conditions used (40), and this, coupled with occasional dissociation provoked by SopB, could account for the heterogeneity of the reaction products.…”

Comparison of biochemical properties of DNA‐topoisomerase I from normal and regenerating liver

“…In addition, topoisomerase I was shown to be inactivated by ADPribosylation (Ferro and Olivera, 1984) and stimulated by interaction with histone H I or high-mobility-group proteins (Javaherian and Liu, 1983). Finally, ATP appears to act as an inhibitor of topoisomerase-I activity (Low and Holden, 1985;Chen and Castora, 1988) although this point is disputed (Goto et al, 1984;Coderoni et al, 1990).Regenerating liver is a refined model for studying in vivo the specific activation of genes and enzymes involved in the cell cycle. Hepatocytes enter the cell cycle synchronously, with a peak of DNA synthesis at 24 h post-hepatectomy and a peak Abbreviations.…”