Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling

Reverse gyrases are atypical topoisomerases present in hyperthermophiles an d are able to positively supercoil a circular DNA. Despite a number of stud ies, the mechanism by which they perform this peculiar activity is still un clear. Sequence data suggested that reverse gyrases are composed of two put ative domains, a helicase-like and a topoisomerase I, usually in a single p olypeptide, Based on these predictions, we have separately expressed the pu tative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli, We show the fo llowing. (i) The full-length recombinant enzyme sustains ATP-dependent posi tive supercoiling as efficiently as the wild type reverse gyrase, (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N -terminal domain and the full-length reverse gyrase, (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C, The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase, (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a struc tural effect upon binding to DNA These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dep endent conformational changes within the whole enzyme, reminiscent of a pro tein motor.