title: Catalytic core of human topoisomerase IIα: insights into enzyme-DNA interactions and drug mechanism.
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.
authors.affiliation: Department of Biochemistry, ‡Department of Pharmacology, and §Department of Medicine (Hematology/Oncology), Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
authors.forename: R Hunter
authors.initials: RH
authors.lastname: Lindsey
authors.forename: MaryJean
authors.initials: M
authors.lastname: Pendleton
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Ashley
authors.forename: Susan L
authors.initials: SL
authors.lastname: Mercer
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Deweese
authors.forename: Neil
authors.initials: N
authors.lastname: Osheroff
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D053837:DNA Cleavage / Q000187:drug effects; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005047:Etoposide / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D059005:Topoisomerase II Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000951:Antigens, Neoplasm; D016227:Benzoquinones; D004270:DNA, Circular; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D059005:Topoisomerase II Inhibitors; C004532:quinone; D005047:Etoposide; D004250:DNA Topoisomerases, Type II; C003466:thymoquinone