Direct observation of DNA overwinding by reverse gyrase

Abstract

Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70°C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71°C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50°C. The unloaded reaction rate at 71°C exceeds five turns per second, which is >10 2 -fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s −1 , indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼5 pN·nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding.reverse gyrase | topoisomerase | magnetic tweezers | DNA overwinding | torsion R everse gyrase, discovered in 1984 in a hyperthermophilic archaeon Sulfolobus (1) which was later classified as Sulfolobus tokodaii (2), is a unique DNA topoisomerase that can introduce positive supercoils into DNA (3-7). The only other enzyme that has the gyration activity is DNA gyrase, which introduces negative supercoils. Although DNA gyrase belongs to type II topoisomerase, which changes the linking number (Lk) of dsDNA by two by cutting and religating both strands simultaneously, reverse gyrase is of type IA topoisomerase (topo IA), where one strand is cut to allow the passage of the other, resulting in the Lk changes in steps of one (3-7). Reverse gyrase is found in hyperthermophilic archaea and eubacteria, and the positive supercoiling activity requires a temperature above 70°C (8, 9). The enzyme is a 130-kDa single polypeptide, a fusion complex of two domains (10): The carboxyl terminal half is related to topo IA, whereas the amino terminal half has an ATP-binding site and is akin to helicase, although neither the whole enzyme nor the isolated helicase-like domain shows genuine helicase activity (11). A crystal structure of the full-length reverse gyrase (12) and more recent structures with additional features (13) all support the basic two-domain construct. The physiological roles of reverse gyrase are not yet fully clear, although positive supercoiling is expected to protect DNA from denaturation at the growth temperatures of hyperthermophiles.Reactions of reverse gyra...

The dynamic interplay between DNA topoisomerases and DNA topology

“…Some Topo IIA (Topo IV, gyrase, and human Topo IIα) are affected by the chirality of DNA Wr supercoiling (Drlica 1992). Reverse gyrase is a unique Type IA topoisomerase combined with a helicase that introduces positive supercoils in an ATP-dependent manner (Ogawa et al 2015;Schoeffler and Berger 2008). The mechanism of positive supercoiling has not been completely established, but it is known that the rate and extent of positive supercoiling are limited by the positive torque on the DNA (Ogawa et al 2015(Ogawa et al , 2016.…”

“…Reverse gyrase is a unique Type IA topoisomerase combined with a helicase that introduces positive supercoils in an ATP-dependent manner (Ogawa et al 2015;Schoeffler and Berger 2008). The mechanism of positive supercoiling has not been completely established, but it is known that the rate and extent of positive supercoiling are limited by the positive torque on the DNA (Ogawa et al 2015(Ogawa et al , 2016. In contrast to Topo IB, the catalytic activities of both Topo IA and IIA decrease with increasing positive torsion, suggesting that the rate-limiting step depends on the torque on the DNA (Charvin et al 2005a;Dekker et al 2002;Ogawa et al 2016;Seol et al 2013a).…”

The dynamic interplay between DNA topoisomerases and DNA topology

“…By working in opposition these two topoisomerases establish a dynamic supercoiling equilibrium that maintains the DNA at a specific level of negative supercoiling (Drlica 1992). Reverse gyrase is a unique type IA topoisomerase combined with a helicase that introduces positive supercoils in an ATP dependent manner (Ogawa et al 2015; Schoeffler and Berger 2008). Whereas the mechanism of positive supercoiling has not been completely established, the rate and extent of positive supercoiling are limited by the positive torque on the DNA (Ogawa et al 2016; Ogawa et al 2015).…”

Torsional stress in DNA limits collaboration among reverse gyrase molecules

“…In a previous study [25], we observed the overwinding reaction under a microscope by tethering a magnetic bead to a coverslip surface with a linear DNA and pulling the bead upward with a magnet. In the presence of reverse gyrase and ATP, the bead rotated continuously to relax the DNA wound by reverse gyrase.…”

“…A limitation of the previous work [25] was that the tension and torsional stress in the DNA could not be independently controlled only by magnetic tweezers, and thus it is still unclear how the torsional stress affects the overwinding rate. In this work we have overcome such limitation by varying the number of working reverse gyrase molecules or the medium viscosity under a constant tension.…”