Abstract
Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.
Highlights
The thermophilic DNA topoisomerase reverse gyrase induces DNA-positive supercoiling
Sequence comparison with bacterial and archaeal Reverse gyrase (RG) shows all conserved motifs typical of the SF2 family helicases in the PcalRG N-terminal domain, and overall high similarity in the C-terminal topoisomerase domain
The protein preparation showed reproducibly, besides a ϳ130 kDa band corresponding to the full-length protein, a ϳ70 kDa protein band corresponding to a truncated product, as shown by its cross-reactivity with an antibody directed against the S. solfataricus TopR1 RG [10]
Summary
The thermophilic DNA topoisomerase reverse gyrase induces DNA-positive supercoiling. Results: The novel reverse gyrase, PcalRG, is presented. The two RG three-dimensional structures available showed overall similarities, but significantly different folds in the N-terminal domain, in particular in the latch and the H1 sub-domain [25, 26] These structural data failed to provide straightforward explanation of the action mechanism as well as the functional differences among RGs. Here, we present a biochemical characterization of the RG from the crenarchaeon Pyrobaculum calidifontis (PcalRG), which appears even more peculiar as compared with other RGs, as it shows: (i) high thermal activity and salt tolerance of its positive DNA supercoiling activity; (ii) the ability of inducing unwinding of synthetic Holliday junctions (HJs); (iii) single strand (ss) DNA annealing; and (iv) strong ssDNA cleavage activity. These features make PcalRG distinct from all other RGs studied so far and reveal additional properties of this class of unconventional enzymes
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