Drugs targeting type II DNA topoisomerases (Top2s) are among the most widely prescribed chemotherapeutic agents for treating lymphomas, leukemias, sarcomas, gastric cancer, testicular cancer, and small cell lung cancer. By interfering with the catalytic cycle of Top2, these drugs exert cell-killing activity by promoting the formation of bulky double-stranded DNA breaks to initiate the cell death pathways. Despite their superior anticancer activities, the prolonged administration of these drugs are known to cause serious side effects, including therapy-related secondary leukemia and cardiotoxicity. Mounting evidences suggest that the undifferentiated drug-targeting of both human Top2 isoforms, hTop2α and hTop2β, is the primary cause of adversity: while targeting of hTop2α is sufficient for killing cancer cells, the hTop2β-induced DNA breaks and chromosome translocation events result in side effects. To overcome these problems, it would be clinically desirable to have a drug that exhibits strong selectivity toward hTop2α. With an established expertise and strength in performing hTop2-related structural analyses, my laboratory have successfully determined the high-resolution crystal structures of clinically active anticancer drugs (mitoxantrone, etoposide, m-AMSA and their derivatives) in complexes with DNA and both hTop2α and hTop2β, which not only revealed the structural basis of drug action but also allowed a set of drug-design guidelines to be formulated. By applying these designing guidelines, a series of novel Pt-derivatized etoposide derivatives (etoplatins) are being developed to preferentially interact with hTop2α by reacting with the α-isoform-specific di-methionine motif. We aim to deliver a new generation of isoform-specific Top2-targeting anticancer drugs upon the completion of this project. In addition to the aforementioned approach, we are also exploring new strategy for interfering with the catalytic cycle of Top2. Mechanistically, Top2 manipulates the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Despite the central importance of this gate-opening event to Top2 function, the DNA-gate in all reported structures of Top2-DNA complexes is in the closed state. By manipulating the crystallization condition, we have obtained the crystal structure of a human Top2 DNA-gate in an open conformation, which not only reveals structural characteristics of its DNA-conducting path, but also uncovers unexpected yet functionally significant conformational changes associated with gate-opening. Using this structure as a starting point, steered molecular dynamics calculations suggest that the Top2-catalyzed DNA passage may be achieved by a rocker-switch-type movement of the DNA-gate. Compounds that bind and arrest Top2 in this newly identified conformational state may exhibit anticancer and antimicrobial activities.