Abstract

Using kinetoplast DNA networks as a substrate in a decatenation assay, we have purified to apparent homogeneity a type I1 DNA topoisomerase from HeLa cell nuclei.The most pure preparations contain a single polypeptide of 172,000 daltons as determined by sodium dodecyl sulfate-gel electrophoresis.The molecular weight of the native protein, based on sedimentation and gel filtration analyses, is estimated to be 309,000.These results suggest that the enzyme is a dimer of 172,000-dalton subunits.The enzyme is a type I1 topoisomerase as demonstrated by its ability to change the linking number of DNA circles in steps of two and to decatenate or unknot covalently closed DNA circles.No gyrase activity is detectable.ATP is required for the relaxation, decatenation, and unknotting of DNA, and a DNA-dependent ATPase activity is present in the most pure fractions.ATP is hydrolyzed to ADP in this reaction.This enzyme is very similar in its catalytic properties to T4 DNA topoisomerase (Liu, L. F., Liu, C.