Locking the ATP-operated clamp of DNA gyrase: Probing the mechanism of strand passage

DNA gyrase catalyses DNA supercoiling by passing one segment of DNA (the T segment) through another (the G segment) in a reaction coupled to the bindi ng and hydrolysis of ATP. The N-terminal domains of the gyrase B dimer cons titute an ATP-operated clamp that is proposed to capture the T segment duri ng the DNA supercoiling reaction. We have locked this clamp in the closed c onformation using the non-hydrolysable ATP analogue ADPNP (5'-adenylyl beta ,gamma -imidodiphosphate). The clamp-locked enzyme is able to bind and clea ve DNA, albeit at a reduced level. Although the locked enzyme is not capabl e of carrying out DNA supercoiling, it can catalyse limited DNA relaxation, consistent with the ability to complete one strand passage event per enzym e molecule via entry of the T segment through the exit gate of the enzyme. The DNA-protein complex of the clamp-locked enzyme has a conformation that differs From the normal positively wrapped conformation of the gyrase-DNA c omplex. These experiments confirm the role of the ATP-operated clamp in the strand-passage reactions of gyrase and suggest a model for the interaction of DNA with gyrase in which a conformation with the T segment in equilibri um across the DNA gate can be achieved via T-segment entry through the ATP- operated clamp or through the exit gate. (C) 2001 Academic Press.