Coupling of adenine nucleotide binding and hydrolysis to single- and double-stranded DNA binding determines the topoisomerase activity of reverse gyrase from 'Thermotoga maritima'

The aim of this PhD thesis was to functionally characterise the hyperthermophilic topoisomerase IA reverse gyrase from the eubacterium Thermotoga maritima with respect to its unique ATP-dependent positive plasmid supercoiling activity. We set out to apply single molecule FRET to observe conformational changes in the N-terminal helicase-like domain and the C-terminal topoisomerase domain of reverse gyrase during topoisomerase activity. Initially, the protocol for the purification of T. maritima reverse gyrase was improved to yield mg amounts of monomeric enzyme with 95% purity. Requirements for positive plasmid supercoiling by reverse gyrase from T. maritima were defined and optimal reaction conditions were established for the rest of the work. However, no universal nucleotide utilisation pattern for topoisomerase activity of reverse gyrases in general exists and various heterogeneous nucleotides-dependent topoisomerase activities have been reported for reverse gyrases from different organisms. Reverse gyrase from T maritima exhibits no topoisomerase activity in the absence of nucleotides but relaxes plasmids in an ADP- and ADPNP-dependent manner. Reverse gyrase positively supercoils plasmid DNA in the presence of ATP. Surprisingly, hydrolysis of ATPγS efficiently promotes positive supercoiling to a similar extend as ATP hydrolysis. Mutation of the GKT sequence in the Walker A motif of the helicase-like domain renders reverse gyrase inactive for nucleotide hydrolysis and reveals vastly reduced topoisomerase activity. Most interestingly, we observed AMP generation in the presence of short double-stranded DNA substrates and plasmid DNA. During plasmid relaxation, ADP and ADPNP are converted into AMP. The same is true for ATP and ATPγS during positive supercoiling. Possibly, ATP is hydrolysed to AMP by reverse gyrase via intermediately generated ADP and energy may be obtained from ADP cleavage. Further investigating the function of the reverse gyrase domains, we demonstrated that the helicase-like domain is a ...