Topoisomerase IV, alone, unknots DNA in E-coli

Knotted DNA has potentially devastating effects on cells. By using two site -specific, recombination systems, we tied all biologically significant simp le DNA knots in Escherichia coli. When topoisomerase IV activity was blocke d, either with a drug or in a temperature-sensitive mutant, the knotted rec ombination intermediates accumulated whether or not gyrase was active. In c ontrast to its decatenation activity, which is strongly affected by DNA sup ercoiling, topoisomerase IV unknotted DNA independently of supercoiling. Th is differential supercoiling effect held true regardless of the relative si zes of the catenanes and knots. Finally, topoisomerase IV unknotted DNA equ ally well when DNA replication was blocked with hydroxyurea. We conclude th at topoisomerase IV, not gyrase, unknots DNA and that it is able to access DNA in the cell freely. With these results, it is now possible to assign co mpletely the topological roles of the topoisomerases in E. coli. It is clea r that the topoisomerases in the cell have distinct and nonoverlapping role s. Consequently, our results suggest Limitations in assigning a physiologic al function to a protein based upon sequence similarity or even upon in vit ro biochemical activity.