Uncoupling of the DNA breaking and rejoining steps of Escherichia coli type I DNA topoisomerase. Demonstration of an active covalent protein-DNA complex.

DNA topoisomerases have been shown to cleave DNA phosphodiester bond and simultaneously become linked to the DNA at the cleavage site via a phosphotyrosine linkage (Tse, Y.-C., Kirkegaard, K., and Wang, J. C. (1980) J. Biol. Chem. 255, 5560-5565). For prokaryotic DNA topoisomerases, this is observed only when denaturant or protease is added to the topoisomerase-DNA incubation mixture. Previous attempts to reform DNA phosphodiester bonds from the covalent protein-DNA complex have been unsuccessful. Using oligonucleotides as substrates, the cleavage reaction of Escherichia coli DNA topoisomerase I occurs spontaneously (Tse-Dinh, Y.-C., McCarron, B. G. H., Arentzen, R., and Chowdhry, V. (1983) Nucleic Acids Res. 11, 8691-8701). Upon reaction with oligo(dA) labeled with 32P using terminal transferase and [alpha-32P]dATP, the enzyme becomes covalently linked to the 32P-labeled oligonucleotide. This 32P label can then be transferred to the 3'-OH end of a linear or nicked duplex DNA molecule subsequently added to the reaction mixture. This phosphodiester bond rejoining reaction can occur at a recessed, blunt, or protruding 3'-end of double-stranded DNA. It requires magnesium ions. These observations suggest that the covalent protein-DNA complex is a true intermediate during topoisomerization. Implications on the structure of prokaryotic type I DNA topoisomerases as compared to their eukaryotic counterparts are discussed.