Reverse gyrase introduces positive supercoils into DNA in an ATP-dependent process. It has a modular structure comprising a helicase-like and a topoisomerase domain. The helicase-like domain consists of two RecA-like subdomains and thus structurally resembles members of the helicase superfamily 2. It is a nucleotide-dependent switch that alters between an ATP state with a slight preference for dsDNA, and an ADP state with a high preference for ssDNA. Inter-domain communication between the helicase-like and the topoisomerase domain is central for their functional cooperation in reverse gyrase. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the nucleotide cycle of the reverse gyrase helicase-like domain in the absence and presence of different DNA substrates. With this comprehensive thermodynamic characterization of the nucleotide cycle of the helicase-like domain, in combination with single molecule FRET data on the conformation of the helicase-like domain at all stages of the catalytic cycle, a picture emerges as to how the helicase-like domain may guide ATP-dependent positive supercoiling by reverse gyrase.