PURIFICATION AND CHARACTERIZATION OF A EUKARYOTIC TYPE-1 TOPOISOMERASE FROM PEA CHLOROPLAST

A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase proper ties are detected in crude lysate of pea chloroplasts using the techni que of transferring P-32 radioactivity from the P-32-labeled DNA to th e protein. The purified enzyme relaxes both positive and negative supe rcoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like c amptothecin and berenil, and unaffected by reagents like novobiocin an d doxorubicin at the topoisomerase II-inhibitory dosage. In the presen ce of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine res idue of the enzyme is responsible for the covalent linkage. Rabbit ant iserum raised against the 16-mer peptide spanning the active residues of hu an topoisomerase I recognizes the 69-kDa protein within the crud e lysate of pea chloroplasts as does the antiserum to the purified 69- kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.