Study On The Interaction Of Mycobacterium Tuberculosis Gyrase B Subunit With DNA

DNA topoisomerase is an indispensable topoisomerase which plays an important role on DNA replication, transcription, and recombination, concerning DNA topology changes. DNA gyrase is the only topoisomerase that is able to introduce negative supercoils into DNA. Mycobacterium tuberculosis gyrase which is the only typeⅡtopoisomerase is a successful target for antibacterial.Four main domains can be found in Mtb, the ATPase domain, the Transducer domain, the Toprim domain and the Tail domain. All truncated mutants were constructed based on the structure of Mtb gyrase B subunit, gyrB1-462, gyrB463-714, gyrB1-631, gyrB1-482, gyrB1-270, gyrB271-480, gyrB485-714, gyrB278-714, gyrB1-542, gyrB1-519 and gyrB1-500, pET28a as its expression vector, and then transformed into E.coli BL21. After expression and purification of wild type GyrB and mutant protein GyrB1-462, GyrB463-714, GyrB1-631, GyrB1-482, GyrB1-270, GyrB271-480, GyrB485-714, GyrB278-714, GyrB1-542, GyrB1-519, GyrB1-500, all the proteins were bind with dsDNA and ssDNA with gel retardation assay. The experiments suggest results as follows. Wild type GyrB has the ability to bind with dsDNA and ssDNA; The deletion of ATPase domain (GyrB278-714) does not affect its binding with dsDNA and ssDNA; The deletion of ATPase domain and Transducer domain (GyrB463-714) has no effect on its binding with dsDNA and ssDNA; The deletion of Toprim domain and Tail domain (GyrB1-462, GyrB1-482) shows little effect on its binding activity with dsDNA and ssDNA; The deletion of C terminal domain (GyrB1-631) has little effect on the binding effect on ssDNA and dsDNA; The deletion of Toprim domain which lays in the CTD of B subunit (GyrBΔtoprim) shows no effect on its dsDNA and ssDNA binding activity. The truncations of single domain of GyrB tend to form inclusion body and can not be expressed or purified. It is suggested that Mtb gyrase B subunit is an ssDNA and dsDNA binding protein, the truncation of the four main domains retains the DNA binding activity, which give a clue that the four domains have different DNA binding activity independently, the deletion of different domain have little effect on its binding activity.