DNA Topoisomerases (topos) are ubiquitous enzymes that catalyze the breakage and rejoining of the DNA phosphodiester backbone, which together with an intervening strand passage event, allow this these enzymes to alter DNA topology (1,2). Intermediates in the strand passage reaction involve either single- or double-stranded breaks defining type I and type II enzymes, respectively. The bacterium Escherichia coli possesses two type I enzymes. One, encoded by the topA gene, is responsible for the major DNA-relaxing activity of the cell and is essential in an otherwise wild-type E. coli (3). E. coli and human topo I (htopoI) both catalyze the relaxation of negatively supercoiled DNA. However, they differ in the details of the reactions they catalyze and share no amino acid sequence homology. E. coli topoI (etopoI) demonstrates a preference for binding at the junction of double- and single-stranded regions, and proceeds using a single 5’-phosphodiester intermediate (4–6). The etopoI enzyme is very efficient at relaxing highly negatively supercoiled DNA and shows progressively decreasing activity as the substrate DNA becomes more relaxed (4,5). The htopoI enzyme relaxes both negatively and positively supercoiled DNA to completion (7). htopoI shows a preference for binding doublestranded DNA and proceeds by making a single covalent 3′-phosphodiester intermediate to a tyrosine (7–9). The htopoI enzyme is the target of the antitumor drug camptothecin (CPT), which traps the covalent phosphotyrosine intermediate of the strand passage reaction (10). The bacterium E. coli and its etopoI enzyme (11) are resistant to CPT.