Final Report Summary - REV GYR MECH (Towards Understanding the mechanism of positive supercoiling by reverse gyrase from Thermotoga maritima)
Reverse gyrase is an unique topoisomerase found in hyperthermophilic organisms that can introduce positive supercoils into DNA in an ATP dependent manner. The enzyme has two closely linked domains: a helicase domain in the N-terminus and a C-terminal type IA topoisomerase domain. With its interesting architecture and unique functionality, the enzyme has lately become subject of interest. Biochemical and biophysical evidences gained so far from different laboratories are not adequate to postulate a well-defined mechanism by which the enzyme introduces topological changes in the DNA. Therefore, we proposed to undertake detailed biophysical studies to gather insight into the mechanistic steps of the enzyme with an attempt to establish a rather feasible model that can link the unique domain architecture to enzyme functioning. Of the several questions we intend to address, the first one we encountered was the mode of DNA and nucleotide binding and the resulting inter-domain communication that facilitates subsequent trans-esterification and supercoiling.
As discussed earlier, the enzyme reverse gyrase is a modular enzyme comprising of a helicase and topoisomerase fused together. Of the two subdomains (H1 and H2), the H2 domain has an insertion called the latch (H3) domain that structurally resembles the RNA binding region of transcription termination factor rho. The latch domain has been speculated to modulate interdomain communication between the helicase and the topoisomerase domain during the supercoiling reaction. We therefore, deleted the latch domain from the whole enzyme and from the helicase domain and observe the effect on supercoiling activity and other major properties of the enzyme. Deletion of latch domain led to loss of co-operativity in nucleotide and DNA binding in the helicase like domain. The latch deficient enzyme also lost the ability to discriminate single and double stranded DNA substrates.
Finally, deletion of the latch domain abolished positive supercoiling activity indicating that the latch domain is indispensible for the positive supercoiling activity. These findings clearly conveys the message that, the latch coordinates individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain. The finding that, the latch domain helps in the initial discrimination of substrate DNA depending on the nucleotide bound state tempted us to test the possibility that the latch associates with the denatured bubble regions on the DNA. Ensemble FRET experiments with fluorescently labelled DNA and proteins indicated the possible proximity of the latch domain to bubble regions on the DNA. Finally, we attempted to capture the domain movements that occur during the catalytic cycle. Based on the only available crystal structure a model describing the probable domain dynamics had been postulated. Since, recent biochemical findings also point towards such a mechanistic model, we chose to validate the same using single molecule FRET.
As, described in this model, upon binding of nucleotide and DNA, there is a conformational change in the helicase domain leading to closure of the domains H1 and H2 with a simultaneous movement of the latch domain away from topoisomerase domain with subsequent swinging up of the lid. Attachment of donor/acceptor fluorescent dyes to the latch and the lid domains followed by measurement of FRET between the dye pair was performed. Our results from this experiment indicate that, the enzyme exhibits a flipping of the domains at sub-physiological temperatures only in presence of large DNA molecules like plasmids and specialised nucleotides. Although the results from the single molecule FRET experiments are rather preliminary, they partly fulfil our objective towards understanding reverse gyrase domain dynamics.
As discussed earlier, the enzyme reverse gyrase is a modular enzyme comprising of a helicase and topoisomerase fused together. Of the two subdomains (H1 and H2), the H2 domain has an insertion called the latch (H3) domain that structurally resembles the RNA binding region of transcription termination factor rho. The latch domain has been speculated to modulate interdomain communication between the helicase and the topoisomerase domain during the supercoiling reaction. We therefore, deleted the latch domain from the whole enzyme and from the helicase domain and observe the effect on supercoiling activity and other major properties of the enzyme. Deletion of latch domain led to loss of co-operativity in nucleotide and DNA binding in the helicase like domain. The latch deficient enzyme also lost the ability to discriminate single and double stranded DNA substrates.
Finally, deletion of the latch domain abolished positive supercoiling activity indicating that the latch domain is indispensible for the positive supercoiling activity. These findings clearly conveys the message that, the latch coordinates individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain. The finding that, the latch domain helps in the initial discrimination of substrate DNA depending on the nucleotide bound state tempted us to test the possibility that the latch associates with the denatured bubble regions on the DNA. Ensemble FRET experiments with fluorescently labelled DNA and proteins indicated the possible proximity of the latch domain to bubble regions on the DNA. Finally, we attempted to capture the domain movements that occur during the catalytic cycle. Based on the only available crystal structure a model describing the probable domain dynamics had been postulated. Since, recent biochemical findings also point towards such a mechanistic model, we chose to validate the same using single molecule FRET.
As, described in this model, upon binding of nucleotide and DNA, there is a conformational change in the helicase domain leading to closure of the domains H1 and H2 with a simultaneous movement of the latch domain away from topoisomerase domain with subsequent swinging up of the lid. Attachment of donor/acceptor fluorescent dyes to the latch and the lid domains followed by measurement of FRET between the dye pair was performed. Our results from this experiment indicate that, the enzyme exhibits a flipping of the domains at sub-physiological temperatures only in presence of large DNA molecules like plasmids and specialised nucleotides. Although the results from the single molecule FRET experiments are rather preliminary, they partly fulfil our objective towards understanding reverse gyrase domain dynamics.