Author/Editor		Golob, Mojca
Title		Kloniranje in izolacija rekombinantnih giraz za razvoj novih giraznih inhibitorjev
Translated title		Cloning and isolation of recombinant gyrases for development of new inhibitors
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta, Biuotehniška fakulteta
Publication year		2001
Volume		str. 124
Language		slo
Abstract		Overuse of antibiotics in humans has led to the rapid evolution of bacteria that are resistant to multiple drugs which cause serious problems in medical treatment of infectious diseases. Because of that the process of drug discovery has been rapidly evolving over the last few years. Modern approach for development of new potential inhibitors is based on the knowledge about structure and funstion of the essential bacterial proteins. Inactivation of this enzyme is lethal to microorganisms. The purpose of our investigations was to establish a full circle of a development of novel antimicrobial drugs-gyrase inhibitors. DNA gyrase, an essential bacterial enzyme belonging to a class of proteins called DNA topoisomerases is one of the most important targets for rational drug design. Gyrase has the ability to catalyse changes in the topology of circular DNA. It differs from topoisomerases class II which have similar function in human. Escherichia coli DNA gyrase, the focus of our work, is composed of two subunits, GyrA (97 kDa) and GyrB (90 kDa). They associate to form an A2B2-active holoenzyme. GyrA protein comprises an Nterminal domain (59-64 kDa), which is responsible for the DNA breakage-reunion and interacts with quinolone group of inhibitors. Other 33 kDa C-terminal is involved in DNAwrapping. GyB subunit also possesses two domains, a 43-kDa N-terminal domain that contains the ATPase catalytic site and interacts with inhibitors (coumarin and cyclotialidine antibiotics). 47-kDa C-terminal domain is required for the interaction with GyrA and DNA. In the first part of our work we have prepared in vitro test system for the evaluation of relaxation and DNA cleavage activity of gyrase caused by quinolone group of inhibitors and supecoiling and inhibition of supecoiling caused by coumarin group of inhibitors. We successfully prepared several miligrams of highly purified and active recombinant gyrase A and B subunits. (Abstract truncated at 2000 characters).
Descriptors		DNA TOPOISOMERASE (ATP-HYDROLYSING) CLONING, MOLECULAR ESCHERICHIA COLI CIPROFLOXACIN COUMARINS PLASMIDS COENZYMES POLYMERASE CHAIN REACTION RECOMBINANT PROTEINS CHROMATOGRAPHY