Separate and Combined Biochemical Activities of the Subunits of a Naturally Split Reverse Gyrase

Abstract

Reverse gyrase reanneals denatured DNA and induces positive supercoils in DNA, an activity that is critical for life at very high temperatures. Positive supercoiling occurs by a poorly understood mechanism involving the coordination of a topoisomerase domain and a helicase-like domain. In the parasitic archaeon Nanoarchaeum equitans, these domains occur as separate subunits. We express the subunits, and characterize them both in isolation and as a heterodimer. Each subunit tightly associates and interacts with the other. The topoisomerase subunit enhances the catalytic specificity of the DNA-dependent ATPase activity of the helicase-like subunit, and the helicase-like subunit inhibits the relaxation activity of the topoisomerase subunit while promoting positive supercoiling. DNA binding preference for both single-and doublestranded DNA is partitioned between the subunits. Based on a sensitive topological shift assay, the binding preference of helicase-like subunit for underwound DNA is modulated by its binding with ATP cofactor. These results provide new insight into the mechanism of positive supercoil induction by reverse gyrase.In the most inhospitable environments, life can survive and even thrive. A striking example of this adaptability is that of hyperthermophilic organisms, which have optimal growth conditions at temperatures of 80°C or higher (1). Life at such extreme temperatures presents unique challenges, which include the propensity for double-stranded DNA to denature and a host of other potential genomic defects (reviewed in Refs. 2 and 3).Hyperthermophiles, whether bacterial or archaeal, deal with the thermal instability of DNA by means of an enzyme called reverse gyrase (reviewed recently in Refs. 4, 5, and 6). Since its discovery in a hyperthermophilic archaeon (7), there is growing evidence suggesting a role of reverse gyrase in stabilizing genomes in hyperthermophiles. It appears to be the only gene specific to hyperthermophiles (8) and is present in all hyperthermophiles and in some thermophiles as well (9).This unique function of reverse gyrase is further supported by genetic evidence. Although reverse gyrase is dispensable for growth of Thermococcus kodakaraensis below 65°C, the strain without reverse gyrase showed retarded growth between 65-90°C, and no growth at 93°C (10), demonstrating the essential role of reverse gyrase in supporting life at extreme temperatures.Reverse gyrase is a type IA topoisomerase and has a unique enzymatic activity in utilizing ATP hydrolysis to induce positive supercoils in DNA. It has not been established precisely how this enzyme can protect against DNA thermal instability. DNA positive supercoiling per se may not be the direct cause, because supercoiling in hyperthermophiles is highly variable ranging from positively supercoiled to relaxed or even negatively supercoiled (11). The biochemical activity of reverse gyrase as a renaturase for single-stranded DNA may have a critical role in maintaining genome stability at high temperature (12). In addition, reverse ...

The Reverse Gyrase from Pyrobaculum calidifontis, a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities

“…The kinetic constants (Fig. 3C) were in the same order of magnitude of those reported for other RGs (13,19,20). Surprisingly, however, steady state ATPase activity of PcalRG deviates from the Michaelis-Menten behavior, showing inhibition of the hydrolysis reaction at ATP concentrations higher than 300 M. Such inhibition has not been reported for other RGs; for instance, the A. fulgidus RG ATPase activity shows hyperbolic dependence on ATP concentration up to 0.3 mM of ATP, and constant velocity from 0.3 to 1.0 mM (15).…”

“…S1). A single putative Zn finger, conserved in all RGs, is present at the N terminus (aa [11][12][13][14][15][16][17][18][19][20]; the region corresponding to the latch is longer than in most of the other RGs (aa 432 to 536), as is the insert in the H1 domain (aa 242-290).…”

“…Sulfolobus RG), retaining their own activities and restoring the positive supercoiling activity when combined. In both enzymes, the C-terminal domain stimulates the N-terminal ATPase activity (13,18,19). In contrast, in Thermotoga maritima RG, each domain was suggested to attenuate the individual activities of the other, although only the N-terminal domain of this enzyme was tested in isolation (20 -22).…”

Strain-level genomic variation in natural populations ofLebetimonasfrom an erupting deep-sea volcano

“…While the genomes of other moderately thermophilic hydrogen-oxidizing groups such as Caminibacter, Nautilia and Nitratiruptor have fulllength reverse gyrase genes, this gene does not appear to confer an advantage to Lebetimonas where reverse gyrase is highly fragmented and lacks the helicase domain. In the parasitic archaeon Nanoarchaeum equitans, the two domains of reverse gyrase are split within the genome, yet they still function together (Capp et al, 2010). A similar scenario may be possible for Lebetimonas; however, no helicase domains homologous to the reverse gyrase-associated helicase in other Nautiliaceae were detected.…”

Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases

“…It has a minimalistic genome of 0.49 Mb and encodes only the absolutely indispensable molecules for life (18,19). The Nanoarchaeum genome codes for two type IA enzymes, reverse gyrase (20) and Top3, both of which are from split genes. Here we show that recombinant Top3 from coexpression of the split genes has the expected type IA enzymatic activities.…”