Characterisation of size variants of type I DNA topoisomerase isolated from calf thymus.
Calf thymus DNA topoisomerase I, which belongs to the eukaryotic type I topoisomerases, is in a typical preparation purified as a set of five major polypeptides with Mr between 70000 and 100000. At least four of these proteins have binding affinity for DNA as was shown by incubating them with radioactive single-stranded DNA after separation in dodecylsulfate polyacrylamide gels and blotting onto nitrocellulose filters. That these polypeptides have DNA relaxing activity was directly demonstrated with protein extracted from single bands of dodecylsulfate/polyacrylamide gels. We consider the 100000-Mr protein to be the native enzyme. The smaller components are catalytically active fragments of the native topoisomerase most probably arising from limited proteolysis either within the nucleus or during the purification of the enzyme. In two-dimensional non-equilibrium pH-gradient electrophoresis gels the topoisomerase size variants exhibit apparent pI values between 8.1 and 8.3, with small but distinct differences between the components. The calf thymus topoisomerase I, upon binding to phage fd-DNA, protects a stretch of 15-25 nucleotides against digestion with DNase I.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D013950:Thymus Gland / Q000201:enzymology
D010446:Peptide Fragments; D004247:DNA; D003850:Deoxyribonuclease I; D004264:DNA Topoisomerases, Type I