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Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cI have been constructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker sequence that connects the N and C-terminal domains of the intact repressor. Compared to the isolated N-terminal DNA-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a slightly stronger repression in vivo. The recognition of the symmetric OR1 operator sequence by this sc homodimer was indistinguishable from that of the naturally dimerized repressor in terms of binding affinity, DNase I protection pattern and in vivo repressor function. Using the new, sc framework, mutant proteins with altered DNA-binding specificity have also been constructed. Substitution of the DNA-contacting amino acid residues of the recognition helix in one of the domains with the corresponding residues of the Salmonella phage P22 repressor c2 resulted in a sc heterodimer of altered specificity. This new heterodimeric molecule recognized an asymmetric, artificial 434-P22 chimeric operator with high affinity. Similar substitutions in both 434 domains have led to a new sc homodimer which showed high affinity binding to a natural, symmetric P22 operator. These findings, supported by both in vitro and in vivo experiments, show that the sc architecture allows for the introduction of independent changes in the binding domains and suggest that this new protein framework could be used to generate new specificities in protein-DNA interaction. # 1997 Academic Press Limited

We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.

The Pvu II restriction endonuclease has been converted from its natural homodimeric form into a single polypeptide chain by tandemly linking the two subunits through a short peptide linker. The arrangement of the single-chain Pvu II (sc Pvu II) is (2-157)-GlySerGlyGly-(2-157), where (2-157) represents the amino acid residues of the enzyme subunit and GlySerGlyGly is the peptide linker. By introducing the corresponding tandem gene into Escherichia coli , Pvu II endonuclease activity could be detected in functional in vivo assays. The sc enzyme was expressed at high level as a soluble protein. The purified enzyme was shown to have the molecular mass expected for the designed sc protein. Based on the DNA cleavage patterns obtained with different substrates, the cleavage specificity of the sc Pvu II is indistinguishable from that of the wild-type (wt) enzyme. The sc enzyme binds specifically to the cognate DNA site under non-catalytic conditions, in the presence of Ca 2+ , with the expected 1:1 stoichiometry. Under standard catalytic conditions, the sc enzyme cleaves simultaneously the two DNA strands in a concerted manner. Steady-state kinetic parameters of DNA cleavage by the sc and wt Pvu II showed that the sc enzyme is a potent, but somewhat less efficient catalyst; the k cat / K M values are 1.11 × 10 9 and 3.50 × 10 9 min −1 M −1 for the sc and wt enzyme, respectively. The activity decrease is due to the lower turnover number and to the lower substrate affinity. The sc arrangement provides a facile route to obtain asymmetrically modified heterodimeric enzymes.