Mechanism of ATP inhibition of mammalian type I DNA topoisomerase: DNA binding, cleavage, and rejoining are insensitive to ATP.
A general, unrefined mechanism of type I DNA topoisomerase action involves several steps including DNA binding, single-strand scission, strand passage resealing, and, possibly, readoption of an active enzyme conformation. None of these steps requires an energy cofactor; however, we have shown previously that several mammalian type I topoisomerases are, in fact, inhibited by ATP. In this study, we wanted to examine which steps in the gross topoisomerase mechanism were sensitive or insensitive to ATP. Nitrocellulose filter binding experiments showed that ATP did not interfere with the binding of DNA by the enzyme and that ATP binding by topoisomerase was 5-fold greater in the presence of DNA than in its absence. Agarose gel electrophoresis in the presence or absence of ethidium bromide indicated that resealing was unaffected by added ATP. The addition of the adenine nucleotide did not alter the pattern of camptothecin-stimulated cleavage of DNA, indicating that strand scission was not the point of inhibition. To test whether strand passage or the readoption of an active conformation was an inhibited step, we used a unique DNA topoisomer as substrate. The results argued against readoption of an active enzyme conformation as an ATP-sensitive process.