ABSTRACT
Coordination between the N-terminal gate and the catalytic core of topoisomerase II allows the proper capture, cleavage, and transport of DNA during the catalytic cycle. Because the activities of these domains are tightly linked, it has been difficult to discern their individual contributions to enzyme-DNA interactions and drug mechanism. To further address the roles of these domains, we analyzed the activity of the catalytic core of human topoisomerase IIα. The catalytic core and the wild-type enzyme both maintained higher levels of cleavage with negatively (as compared to positively) supercoiled plasmid, indicating that the ability to distinguish supercoil handedness is embedded within the catalytic core. However, the catalytic core alone displayed little ability to cleave DNA substrates that did not intrinsically provide the enzyme with a transport segment (i.e., substrates that did not contain crossovers). Finally, in contrast to interfacial topoisomerase II poisons, covalent poisons did not enhance DNA cleavage mediated by the catalytic core. This distinction allowed us to further characterize the mechanism of etoposide quinone, a drug metabolite that functions primarily as a covalent poison. Etoposide quinone retained some ability to enhance DNA cleavage mediated by the catalytic core, indicating that it still can function as an interfacial poison. These results further define the distinct contributions of the N-terminal gate and the catalytic core to topoisomerase II function. The catalytic core senses the handedness of DNA supercoils during cleavage, while the N-terminal gate is critical for capturing the transport segment and for the activity of covalent poisons.
Subject(s)
Antigens, Neoplasm/metabolism , DNA Topoisomerases, Type II/metabolism , DNA, Circular/metabolism , DNA, Superhelical/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Models, Molecular , Topoisomerase II Inhibitors/pharmacology , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Benzoquinones/chemistry , Benzoquinones/metabolism , Benzoquinones/pharmacology , Binding Sites , Biocatalysis/drug effects , Catalytic Domain , DNA Cleavage/drug effects , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , DNA, Circular/chemistry , DNA, Superhelical/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Etoposide/chemistry , Etoposide/metabolism , Etoposide/pharmacology , Humans , Kinetics , Molecular Conformation , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Stereoisomerism , Substrate Specificity , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/metabolismABSTRACT
Type II topoisomerases are essential enzymes that modulate DNA under- and overwinding, knotting, and tangling. Beyond their critical physiological functions, these enzymes are the targets for some of the most widely prescribed anticancer drugs (topoisomerase II poisons) in clinical use. Topoisomerase II poisons kill cells by increasing levels of covalent enzyme-cleaved DNA complexes that are normal reaction intermediates. Drugs such as etoposide, doxorubicin, and mitoxantrone are frontline therapies for a variety of solid tumors and hematological malignancies. Unfortunately, their use also is associated with the development of specific leukemias. Regimens that include etoposide or doxorubicin are linked to the occurrence of acute myeloid leukemias that feature rearrangements at chromosomal band 11q23. Similar rearrangements are seen in infant leukemias and are associated with gestational diets that are high in naturally occurring topoisomerase II-active compounds. Finally, regimens that include mitoxantrone and epirubicin are linked to acute promyelocytic leukemias that feature t(15;17) rearrangements. The first part of this article will focus on type II topoisomerases and describe the mechanism of enzyme and drug action. The second part will discuss how topoisomerase II poisons trigger chromosomal breaks that lead to leukemia and potential approaches for dissociating the actions of drugs from their leukemogenic potential.
