Study On The Interaction Of Mycobacterium Tuberculosis Gyrase With DNA

As it is exclusively present in prokaryotes and is essential for cell survival, DNA gyrase (EC 5.99.1.3) is potential targets of antibacterial and antineoplastic drugs. The discoveries about gyrase have led to the expansion of interest in its basic and clinical research. As an infectious disease caused by Mycobacterium tuberculosis and leading cause of deaths resulting from a single infectious disease, tuberculosis is on the rise and is revisiting in the world, especially in China, and becomes a worldwide and aggravated problem. DNA gyrase is involved in crucial cellular processes of M. tuberculosis, and is one of main factors causing the emergence of drug-resistant strains. As only a typeâ…¡topoisomerase existing in the bacillus, it is greatly important to study on M. tuberculosis DNA gyrase.Through biology informatics softwares, molecular cloning, gel-retardation electrophoresis and surface plasmon resonance et al, the results showed that the gyrA and gyrB of M. tuberculosis DNA gyrase have high content of GC bases and there are many biased amino acids composing of their coding proteins. The reconstituted M. tuberculosis DNA gyrase exhibits the proper enzymic activities, and appears the highest supercoiling activity under proper conditions such as 30℃of reaction temperature. It is proved that M. tuberculosis GyrB is an ssDNA-binding protein rather than a dsDNA-binding protein, the combined A₂B₂ and GyrA both are a dsDNA- and ssDNA-binding protein and have a strong DNA-binding activity, but the GyrA-NTD and GyrA-CTD only bind to the dsDNA with specific sites. It is interesting that binding DNA of M. tuberculosis DNA gyrase subunits and combined holoenzyme are all independent on Mg²⁺. The DNA-binding conditions of M. tuberculosis GyrA, such as binding temperature, were optimized. However, Ca²⁺, EDTA and norfloxcin both with proper concentration, hinders M. tuberculosis GyrA to bind dsDNA. The DNA-binding activity of M. tuberculosis GyrA is homologous to those of Pyrobest Polymerase, BSA et al. Furthermore, M. tuberculosis GyrA can bind different topological DNA, and can bind the least length of 36-bp DNA. The results also showed that Y577, R691 and R745 are among the key DNA-binding residues in M. tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M. tuberculosis DNA gyrase, so the M. tuberculosis GyrA-CTD is taken for a potential new target for drug design. It was found that the quinolone-resistant-double-mutation of M. tuberculosis GyrA or combined A₂B₂ have a higher competence of binding ssDNA and dsDNA than those of wild-type, and norfloxcin can hinder M. tuberculosis GyrA or combined A₂B₂ to bind ssDNA and dsDNA. Based on it, the possible drug-resistant mechanism of M. tuberculosis and the possible restrained-bacterium mechanism of norfloxcin were presented, respectively.The DNA-binding activity of GyrB, the DNA-binding sites in GyrA-CTD, the effect of GyrA-CTD on relaxation activity and the double-mutation with a higher DNA-binding capacity, are all the first time to have been found. It is helpful to make clear the DNA-binding mechanism and reaction mechanism of DNA gyrase, to explain the biological characteristic and pathogenic mechanism of M. tuberculosis, and to provide new targets for drug design.