Biochemical characterization of topoisomerase I purified from avian erythrocytes

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Abstract
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 P-DNA, digesting with nuclease and detecting 32-p labeled peptides by sodium dodecyl sulfate polyaorylamide gel electrophoresis. Topoisomerase activity, as measured by the ability to oovalently bind DMA, 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. Nat 1. Acad. Soi. USA 78, 3487–3191). 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 micrococoal 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.