Biochemical characterization of topoisomerase I purified from avian erythrocytes.

A type I topoisomerase has been purified from avian erythrocyte nuclei. The most pure fraction contains one major polypeptide of Mr = 105,000 (80% of total) and several minor ones of lower molecular weight. Active forms of the topoisomerase were identified by covalently binding the enzyme to 32P-DNA, digesting with nuclease and detecting 32P labeled peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Topoisomerase activity, as measured by the ability to covalently bind DNA, is associated with the following peptides: Mr = 105, 83, 54 and 30,000. The similar chromatographic properties of the various forms of topoisomerase suggests a common structural identity as previously proposed for the HeLa topoisomerase I (Liu, L.F. and Miller, K.G. (1981) Proc. Natl. Acad. Sci. USA 78, 3487-3491). The avian enzyme is similar to other eucaryotic type I DNA topoisomerases in that it covalently binds double and single stranded DNA forming an enzyme linked to the 3'-phosphoryl end and after binding to single stranded DNA it can transfer the single stranded donor DNA to an acceptor DNA possessing 5'-OH end groups. The binding site size of topoisomerase on DNA has also been determined using micrococcal nuclease to digest unprotected DNA in the native enzyme/DNA complex. The enzyme blocks access to the helix over a span of 25 bp. These findings are discussed in light of the distribution and function of topoisomerase I in chromatin.