Energy Coupling in Escherichia coli DNA Gyrase: the Relationship between Nucleotide Binding, Strand Passage, and DNA Supercoiling † Andrew D. Bates,* ,‡ Mary H. O’Dea, § and Martin Gellert § Department of Biochemistry, UniVersity of LiVerpool, P.O. Box 147, LiVerpool, L69 3BX, U.K., and Laboratory of Molecular Biology, National Institute of Diabetes and DigestiVe and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892 ReceiVed October 11, 1995; ReVised Manuscript ReceiVed NoVember 27, 1995 X ABSTRACT: Binding of the nonhydrolyzable ATP analogue 5′-adenylyl-,γ-imidodiphosphate (ADPNP) to Escherichia coli DNA gyrase can lead to a limited noncatalytic supercoiling of DNA. Here we examine the efficiency of coupling between ADPNP binding and the change in linking number either of positively or negatively supercoiled plasmid DNA or of small DNA circles. The coupling efficiency varies from 100% (∆Lk )-2 per gyrase tetramer, a stoichiometry of 1) with positively supercoiled substrates under certain reaction conditions to an undetectably low value with moderately negatively supercoiled substrates (σ )-0.046) or small circular substrates. Furthermore, the rate of ADPNP binding to the gyrase-DNA complex is also dependent on the topological state of the DNA; the previously observed slow binding of ADPNP to the complex of gyrase with linear DNA is accelerated 16-fold when the substrate DNA is negatively supercoiled, suggesting a functional interaction between the nucleotide-binding and DNA- binding domains which is independent of the strand-passage process. The implications for the normal ATP-dependent supercoiling reaction of the enzyme are considered and the results discussed in terms of current mechanistic models for DNA gyrase action and the possible in ViVo roles of the enzyme. DNA gyrase is unique among the type II DNA topo- isomerase enzymes in its ability to utilize the free energy of ATP hydrolysis for the introduction of negative supercoiling into closed-circular DNA. All other type II enzymes are able only to relax supercoiling already present in DNA. Gyrase provides an accessible system exemplifying energy transduction in biological processes (Reece & Maxwell, 1991). Active Escherichia coli DNA gyrase is a tetramer of two A and two B subunits, of 97 and 90 kDa, respectively. The tetramer binds to double-stranded DNA, with approximately 130 base pairs (bp) 1 of DNA being wrapped around the complex in a positive superhelical sense (Orphanides & Maxwell, 1994). The DNA is cleaved in both strands, roughly in the center of the wrapped segment, via phos- phodiester exchange with the Tyr-122 residues of the A subunits, leading to covalent attachment of the 5′-ends of the cleaved strands to the protein. Another segment of DNA, which in the supercoiling reaction is probably near or within the wrapped region, is then passed through the double- stranded break, resulting in the reduction of the linking number (Lk) of a closed-circular DNA by two. The strands are then resealed by the reverse exchange reaction. At some point in this process ATP is hydrolyzed by the B subunits, providing energy for the incorporation of torsional stress into the DNA molecule. The exact mechanism of the coupling of ATP hydrolysis to the introduction of supercoiling is not known in detail, although the ability of the nonhydrolyzable ATP analogue 5′-adenylyl-,γ-imidodiphosphate (ADPNP) to promote limited noncatalytic supercoiling of relaxed DNA by gyrase has led to the suggestion that the initial binding of nucleotide causes the translocation of DNA through the double-stranded break (i.e., the supercoiling event) and that hydrolysis and product release are responsible for the cycling of the complex to allow catalytic supercoiling (Sugino et al., 1978). It has been suggested in the case of the homologous eukaryotic enzyme, topoisomerase II, that the strand-passage process proceeds by the trapping of the translocated strand through the closing of a nucleotide- operated clamp (Roca & Wang, 1992). Gyrase can also relax positive supercoils, in an ATP-dependent reaction essentially equivalent to the introduction of negative supercoils, and relax negative supercoils in a nucleotide-independent fashion. A variety of evidence suggests that two molecules of ATP are hydrolyzed per supercoiling cycle. The assumption that one nucleotide molecule binds to each B subunit has been confirmed from studies of the binding of nucleotide ana- logues to the gyrase-DNA complex (Tamura et al., 1992; Tamura & Gellert, 1990). In addition, the X-ray-derived structure of crystals of the N-terminal 43 kDa portion of the B subunit in the presence of ADPNP has revealed a dimer containing one molecule of the ATP analogue per monomer (Wigley et al., 1991). It has been shown that the supercoiling of a plasmid DNA substrate by gyrase eventually reaches a limit corresponding to a specific linking difference (σ) of -0.11 (Bates & Maxwell, 1989; Westerhoff et al., 1988). This corresponds to a reduction in the linking number of pBR322 DNA (length N ) 4361 bp) of ∼46. The free † Work in A.D.B.’s laboratory was funded by the Wellcome Trust (Grant No. 039713/z/93). * Corresponding author. Telephone: (44) 151 794 4322. Fax: (44) 151 794 4349. E-mail: bates@liv.ac.uk. ‡ University of Liverpool. § NIH. X Abstract published in AdVance ACS Abstracts, January 1, 1996. 1 Abbreviations: bp, base pairs; Lk, linking number; ADPNP, 5′- adenylyl-,γ-imidodiphosphate; σ, specific linking difference; N, length of DNA in base pairs; EtBr, ethidium bromide; ∆Lk, linking difference; GyrA, gyrase A protein; GyrB, gyrase B protein; DTT, dithiothreitol; BSA, bovine serum albumin; oc, open-circular (nicked) DNA. 1408 Biochemistry 1996, 35, 1408-1416 0006-2960/96/0435-1408$12.00/0 © 1996 American Chemical Society