Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop

Using heteroduplex molecules formed from a pair of plasmids, one of which contains a small deletion relative to the other, it is shown that bacterial topoisomerase I can relax a positively supercoiled DNA if a short single-stranded loop is placed in the DNA. This result supports the postulate that the specificity of bacterial DNA topoisomerase I for negatively supercoiled DNA in its relaxation reaction derives from the requirement of a short single-stranded DNA segment in the active enzyme-substrate complex. Nucleolytic and chemical probing of complexes between bacterial DNA topoisomerase I and heteroduplex DNA molecules containing single-stranded loops ranging from 13 to 27 nucleotides in length suggests that the enzyme binds specifically to the region containing a single-stranded loop; the site of DNA cleavage by the topoisomerase appears to lie within the single-stranded loop, with the enzyme interacting with nucleotides on both sides of the point of cleavage.