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The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.

The observation that a cis-acting DNA sequence can be separated from its site of action by long distances has been made in many different biological systems, but is not yet understood at the molecular level (for reviews, see Dynan and Tjian 1985;Ptashne 1986). The Hin sitespecific recombination system found in Salmonella typhimurium provides a simple model for understanding this phenomenon. The Hin protein mediates a site-specific DNA inversion that is promoted by the presence of a DNA sequence, a recombinational enhancer, which acts in an orientation-and distance-independent manner (Johnson and Simon 1985). Elucidation of the mechanism of action of the recombinational enhancer may help our understanding of the function of the large variety of enhancer sequences found in other systems.Inversion of the 996-bp DNA segment by Hin alters the expression of flagellin type in Salmonella (Zieg et al. 1977). The recombination reaction requires three DNA sequences: two recombination sites, hixL and hixR, which mark the boundary of the inversion sequence , and the recombinational enhancer (Johnson et al. 1986). Each recombination site is composed of two half-sites with approximate dyad symmetry. The hix sites share a common sequence which must be in inverted configuration for recombination to 3Current address:

A synthetic 52-residue peptide based on the sequence-specific DNA-binding domain of Hin recombinase (139-190) has been equipped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. In the presence of Fe(II), this synthetic EDTA-peptide cleaves DNA at Hin recombination sites. The cleavage data reveal that the amino terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage.

The site‐specific inversion reaction controlling flagellin synthesis in Salmonella involves the function of three proteins: Hin, Fis and HU. The DNA substrate must be supercoiled and contain a recombinational enhancer sequence in addition to the two recombination sites. Using mutant substrates or modified reaction conditions, large amounts of complexes can be generated which are recognized by double‐stranded breaks within both recombination sites upon quenching. The cleaved molecules contain 2‐bp staggered cuts within the central dinucleotide of the recombination site. Hin is covalently associated with the 5′ end while the protruding 3′ end contains a free hydoxyl. We demonstrate that complexes generated in the presence of an active enhancer are intermediates that have advanced past the major rate limiting step(s) of the reaction. In the absence of a functional enhancer, Hin is also able to assemble and catalyze site‐specific cleavages within the two recombination sites. However, these complexes are kinetically distinct from the complexes assembled with a functional enhancer and cannot generate inversion without an active enhancer. The results suggest that strand exchange leading to inversion is mediated by double‐stranded cleavage of DNA at both recombination sites followed by the rotation of strands to position the DNA into the recombinant configuration. The role of the enhancer and DNA supercoiling in these reactions is discussed.