ESCHERICHIA-COLI DNA TOPOISOMERASE-III IS A SITE-SPECIFIC DNA-BINDINGPROTEIN THAT BINDS ASYMMETRICALLY TO ITS CLEAVAGE SITE

The binding of DNA topoisomerase III (Topo III) to a single-stranded D NA substrate containing a strong cleavage site has been examined. The minimal substrate requirement for Topo III-catalyzed cleavage has been determined to consist of 7 bases: 6 bases 5' to the cleavage site and only 1 base 3' to the site. Nuclease P1 protection experiments indica te that the enzyme also binds to its substrate asymmetrically, protect ing similar to 12 bases 5' to the cleavage site and only 2 bases 3' to the cleavage site. A catalytically inactive mutant of Topo III shows the same protection pattern as the active polypeptide, indicating that Topo III is a site-specific binding protein as well as a topoisomeras e. Consistent with this view, an oligonucleotide containing a cleavage site is a more effective inhibitor and is bound more efficiently by T opo III than an oligonucleotide without a cleavage site.