DNA Gyrase And Topo NM From Mycobacteria : Insights into Mechanism And Drug Action

Abstract

Maintenance of a topological homeostasis by introduction and removal of the supercoils to relieve excessive strain on the DNA is a hallmark of topoisomerase function in the cell. The requirement of the topoisomerases during DNA transaction processes marks a ubiquitous presence of the enzymes in all the life forms. Different reactions carried out by the enzymes include relaxation of positive and negative supercoils required majorly during DNA replication and transcription, decatenation at the end of DNA replication to separate the daughter chromosomes and removal of lethal knots generated in the circular chromosome. In eubacteria, the enzymes introduce negative supercoils to facilitate easier strand separation for DNA transaction processes. However, in thermophiles, a different enzyme maintains the genome in a positively supercoiled form to protect from denaturation by excessive heat. These varied functions are carried out by different topoisomerases. Therefore, each organism maintains a minimum required set of the enzymes and the absence of a certain enzyme may be compensated for by topoisomerases with dual functions. For example, Mycobacterium tuberculosis and many other slow growing mycobacteria do not possess topoisomerase IV or its homologs. In these organisms, the DNA gyrase is suggested to carry out both negative supercoiling and decatenation reactions. Therefore, the mycobacterial DNA gyrase must be able to manage between both the functions in vivo. In contrast, Mycobacterium smegmatis and few other mycobacteria contain an additional type II topoisomerase which does not resemble any known type II enzyme but could catalyze relaxation and decatenation reactions. Importantly, the enzyme displays a unique ability to introduce limited positive supercoils and may have certain functions inside the cell which remains to be studied. Owing to the indispensability for bacterial survival topoisomerases present themselves as important drug targets. A large number of inhibitors have been found to inhibit the enzyme and thereby killing the bacterial. Among these, quinolones are successfully being used as broad spectrum antibacterial drugs. Although the commonly used quinolones inhibit many bacterial pathogens, a reduced susceptibility is exhibited by some of the pathogens e.g. Mycobacterium tuberculosis. To circumvent the lower efficacy of existing drugs, new and modified quinolones have been developed which are highly effective against mycobacteria. The difference in the susceptibility may be conferred by a difference in the chemical property of the drug and the interacting residues present in the enzyme. In the present thesis efforts have been made to understand the mechanism of the type II topoisomerases from mycobacteria and drug action on these enzymes. The thesis is divided into four chapters. In Chapter I of the thesis an introduction is provided on the topoisomerases, their classification and different reactions catalyzed by these enzymes. As the work in present thesis has been carried out with type II topoisomerases, introduction of type II enzymes, their structure and mechanisms is elaborated. DNA gyrase, its mechanism of reaction and in vitro and in vivo functions are explained in great detail. DNA gyrase and topoisomerase IV are targeted by a range of different inhibitors. These different classes of inhibitors and their mechanism of action are described. Finally, the mechanism of mycobacterial DNA gyrase with structural information and the current understanding of quinolone action on the enzyme are explained. The chapter ends with the objective of the study in the present thesis. In chapter II, the studies are aimed at understanding the molecular basis for decatenation carried out by mycobacterial DNA gyrase. Previous work from the laboratory showed that the enzyme can carry out decatenation more efficiently than its homolog from E. coli. It was shown that the mycobacterial enzyme binds two DNA molecules in trans in a length dependent manner. The ability to bind the second DNA is conferred upon the holoenzyme by ATPase subunit (GyrB) subunit which alone can bind DNA. Similar studies using topo IV from E. coli, the strongest known decatenase showed binding of two DNA molecules and the second DNA binding by ATPase (ParE) subunit. However, GyrB subunit from E. coli DNA gyrase, a weaker decatenase, does not bind second DNA molecule efficiently. The results provide a general mechanism for decatenation by type II enzymes in which efficient binding of second DNA is important. In Chapter III, studies have been carried out using topo NM, an atypical type II topoisomerase from Mycobacterium smegmatis. The enzyme has been characterized previously in the laboratory. In addition to efficient decatenation and relaxation, the enzyme exhibits a unique ability to introduce positive supercoils into the DNA. As demonstrated for the mycobacterial DNA gyrase and topo IV in the Chapter II, the ATPase subunit (Topo N) of topo NM, binds second DNA efficiently. The binding of both gate and transport segments increases with the length of the DNA. Binding of two DNA molecules by the holoenzyme appears to be a cumulative effect of DNA binding to individual subunits. In the absence of any inhibitor, the enzyme accumulates cleaved DNA products with shorter DNA but not with larger DNA. The cleavage of the shorter DNA is supported only in the presence of Mg2+ and Mn2+. Another important property of the enzyme is to introduce positive supercoils which appears to be due to its efficient utilization of ATP and a high rate of reaction. Chapter IV deals with the interaction of mycobacterial gyrase with fluoroquinolones (FQs). Although DNA gyrase is the sole target of the FQs in M. tuberculosis, the lower susceptibility to commonly used FQs have led to the studies to find out more effective quinolones. Previous studies from the laboratory showed a lower susceptibility of the mycobacterial gyrase to ciprofloxacin, but moxifloxacin could inhibit the enzyme efficiently. The better inhibition by moxifloxacin appears to be due to efficient trapping of the enzyme-DNA covalent complex. Both ciprofloxacin and moxifloxacin bind the DNA gyrase from mycobacteria, E. coli and E. coli topo IV, independent of DNA. The extent of binding also correlates with the inhibition potential of the drug against a given enzyme. A general model of quinolone enzyme interaction is provided wherein the quinolones are shown to interact with GyrA subunit or holoenzyme or the enzyme- DNA complex which would finally result in the trapping of the covalent complex.