Basis for the Discrimination of Supercoil Handedness During DNA Cleavage by Human and Bacterial Type II Topoisomerases

Abstract

Type II topoisomerases are essential enzymes that regulate the topological state of DNA. These enzymes function by performing a double-stranded DNA passage reaction, the process of which generates a covalent enzyme-cleaved DNA complex (i.e., cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. During biological processes such as DNA replication and transcription, cleavage complexes that are formed with positively-supercoiled [(+)SC] DNA are at greater risk of being converted to nonligatable genomic lesions. Consequently, the potential lethality of cleavage complexes makes type II topoisomerases targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled [(-)SC] over (+)SC DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness during DNA cleavage. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage is not fully understood. This dissertation describes how the basis for supercoil recognition by type II topoisomerases during DNA cleavage was determined. A series of benchtop and rapid-quench flow kinetics experiments was carried out that examined the individual steps of DNA cleavage, including persistence, religation, and forward rates of cleavage. Our data demonstrate that the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIβ, gyrase, and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. In the absence of drugs, cleavage complexes are substantially less stable and rapidly fall apart, even if supercoil recognition during cleavage is observed. Finally, rates of enzyme-mediated DNA religation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide at least a partial basis for how type II topoisomerases recognize their DNA substrates.