Dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine †

Abstract

Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.

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

“…The relative preference of single-stranded DNA is reduced in the presence of ADP when compared with the conditions without cofactor or with ATP analog. On the other hand, the engineered recombinant helicase-like domain from the Sulfolobus solfataricus enzyme showed no nucleotide-modulated shift in DNA binding affinity by mobility shift assays (26), similar to our observations reported here. The complicated difference in the binding preferences observed in these experiments could be due to methodology for monitoring binding reactions or to the use of reverse gyrases from different species.…”

Synergic and Opposing Activities of Thermophilic RecQ-like Helicase and Topoisomerase 3 Proteins in Holliday Junction Processing and Replication Fork Stabilization

“…S2). Although the N-terminal domain is an ATPase, but not a processive helicase (34), both Hel112 and Hel308/Hjm were reported to induce duplex unwinding (15)(16)(17)(18), but also ssDNA annealing (17,18), a feature shared by several eukaryotic RecQ homologs (2,5).…”

“…In such assays, co-incubation of E. coli RecQ and Top3 catalyzes the relaxation of negatively supercoiled DNA molecules (6). In contrast, the S. solfataricus reverse gyrase TopR1, as well as the combination of its isolated helicase-like domain (N-terminal) and topoisomerase domain (C-terminal), induce positive supercoiling (34). Because this latter reaction requires high temperature, in 1 and 2) or the same concentration of BSA for mock treatment (lanes 3 and 4).…”

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

“…6.5, 150 mM NaCl, 20% glycerol). Recombinant His-tagged S. solfataricus TopR1 reverse gyrase (hereafter called RG) as well as deletion and site-specific mutants were purified as described previously (21).…”

“…In addition, exploiting the cleavagereligation activity of its topoisomerase domain (21), RG might also act as recombinases, namely by cleavage, exchange, and religation of two DNA strands. We thus sought to elucidate the HJ processing reaction mechanism.…”