Arabidopsis thaliana DNA gyrase: expression, characterisation and in vivo insight

DNA gyrase is a type II topoisomerase distinguished by its ability to introduce negative supercoils into double-stranded DNA in a reaction linked to ATP hydrolysis. The essentiality of gyrase in bacteria has permitted its exploitation as an antibacterial target. The unanticipated discovery of gyrase within the nuclear genomes of eukaryotes including Arabidopsis and Plasmodia, was made near to two decades ago. Despite this, our understanding of gyrase within these species remains limited. The work here aimed to heterologously generate eukaryotic gyrases in order to biochemically characterise and better understand their mechanism of actions, gain an insight into their in vivo functions and explore their potential for inhibition. The specific inhibition of gyrase within these species would facilitate the generation of novel herbicidal and antimalarial drugs. In vivo knockdown experiments of A. thaliana gyrase have confirmed the embryo-lethality of GyrA. Arabidopsis plants able to propagate with a knockdown of GyrB1 are dwarfed, chlorotic, have reduced numbers and lengths of lateral roots and altered thylakoid ultrastructure. An increase of GyrB1 transcript mediates a stress response within Arabidopsis. The functional cooperation to achieve supercoiling of a reconstituted gyrase comprising A. thaliana GyrA and E. coli GyrB has been shown. The catalysis of A. thaliana enzyme (GyrA and GyrB2) is differentially mediated by potassium glutamate levels. The A. thaliana DNA gyrase has been determined to be 45-fold more efficient for ATP-independent DNA relaxation than E. coli gyrase. A novel sensitive DNA decatenation substrate, ‘bis-cat’, comprising two singly-linked supercoiled plasmids of disparate sizes has been generated and compared to the current marketed decatenation substrate. The novel substrate determined A. thaliana gyrase to be 35-fold more effective for DNA decatenation than the E. coli enzyme. The herbicidal and bactericidal specificities of novel fluoroquinolone compounds have also been compared