The Reverse Gyrase from Pyrobaculum calidifontis,a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities |
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Authors: | Anmbreen Jamroze Giuseppe Perugino Anna Valenti Naeem Rashid Mosè Rossi Muhammad Akhtar Maria Ciaramella |
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Institution: | From the ‡Institute of Protein Biochemistry and ;¶Institute of Biosciences and Bioresources, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131, Naples, Italy and ;the §School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan |
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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. |
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Keywords: | DNA Enzymes DNA Helicase DNA Recombination DNA Repair DNA Topoisomerase Holliday Junction Thermophiles |
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