Topoisomerase I (TOP-I) is an essential eukaryotic enzyme that acts to remove supercoils generated during transcription and DNA replication (1). Because of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. Being a type 1 enzyme, TOP-I mediates DNA relaxation by creating a transient, single-strand break in one strand of the DNA duplex. This transient nicking allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr723 in the human TOP-I) at a DNA phosphodiester bond forming a 3′-phosphotyrosine covalent enzyme-DNA complex. The covalent intermediate is reversed when the released 5′-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction (1). The rate of relegation is normally much faster than is the rate of cleavage (2). This ensures that the steady state concentration of the covalent 3′-phosphotyrosyl TOP-I-DNA complex is extremely low. Several DNA lesions and drugs, however, have been shown to stabilize the covalent 3′-phosphotyrosyl intermediate (3). For example, camptothecin (CPT) is a natural product that was originally discovered because of its antitumor activity (4) and was later demonstrated to promote the accumulation of TOP-I-DNA adducts in vitro and in vivo (5,6). It is generally believed that CPTs act to convert TOP-I into a DNA-damaging agent by binding the covalent 3′-phosphotyrosyl intermediate and, specifically, blocking DNA relegation (7, 8). Topo I is the sole intramolecular target of CPT and the cytotoxic effects of CPT poisoning are S-phase-specific (9). Both in vitro and in vivo data support the idea that during DNA replication, the replication complex can collide with the “trapped” TOP-I-DNA complex, resulting in a double-strand break and subsequent apoptotic cell death (10). Presumably, these compounds have anticancer activity because rapidly dividing cells (e.g., cancer cells) enter S-phase more frequently than do normal cells.