BINDING OF ESCHERICHIA-COLI PRIMARY REPLICATIVE HELICASE DNAB PROTEINTO SINGLE-STRANDED-DNA - LONG-RANGE ALLOSTERIC CONFORMATIONAL-CHANGESWITHIN THE PROTEIN HEXAMER

Quantitative analyses of the interactions of the Escherichia coli prim ary replicative helicase DnaB protein with single-stranded ssDNA have been performed using the thermodynamically rigorous fluorescence titra tion technique, This approach allowed us to obtain absolute stoichiome tries of the formed complexes and interaction parameters? without any assumptions about the relationship between the observed signal change and the degree of binding. The analysis of the DnaB helicase interacti ons with nonfluorescent, unmodified nucleic acids has been performed, using a novel spectroscopic Macromolecular Competition Titration (MCT) method developed in the accompanying paper [Jezewska, M. J., & Bujalo wski, W. (1996) Biochemistry, 35, 2117-2128]. In the presence of the A TP nonhydrolyzable analog AMP-FNP, the DnaB helicase binds polymer ssD NA with a site-size of 20 +/- 3 nucleotides per protein hexamer. This site-size is independent of the type of nucleic acid base as well as t he salt concentration and type of salt. Direct thermodynamic studies o f the polynucleotide and oligomer binding to the DnaB hexamer, as well as the competition studies, show that independently of the type of nu cleic acid base, as well as salt concentration and type of salt in sol ution, the helicase has only a single, strong binding site for DNA. On ly this site is used when the protein interacts with polymer ssDNA. Mo reover, UV photo-cross-linking experiments with oligonucleotides of di fferent lengths, dT(pT)(19), dT(pT)(55), and dT(pT)(69), suggest that primarily a single subunit of the DnaB helicase hexamer is in contact with the ssDNA. In interactions with polymer nucleic acids, the DnaB p rotein shows preferential intrinsic affinity for poly(dA), characteriz ed in our standard conditions (pH 8.1, 10 degrees C, 100 mM NaCl, 5 mM MgCl2) by the intrinsic binding constant K = 6 +/- 2 x 10(6) M(-1). T hese affinities are comparable to the affinities of the single-strand binding proteins in the corresponding solution conditions and strongly suggest that the helicase is capable of binding ssDNA without additio nal facilitating factors. Both the intrinsic affinity and the cooperat ivity are salt dependent. The formation of the DnaB-ssDNA complex is a ccompanied by the net release of similar to 2 ions, while another net release of similar to 2 ions accompanies the cooperative interactions. The data indicate an anion effect on the studied interactions and sug gests that the released ions most probably originate from both the pro tein and the nucleic acid. The presence of a single, strong binding si te on the hexamer, built of six chemically identical subunits, the ver y low site-size of the large helicase-ssDNA complex, and the involveme nt of a single subunit in contact with the nucleic acid indicate the p resence of long-range allosteric interactions in the DnaB helicase whi ch encompass the entire DnaB hexamer, Our sedimentation velocity measu rements of the DnaB protein-(AMP-PNP)-5'-fluorescein-(dT)(20) ternary complex show that the sedimentation coefficient of the complex is s(20 ,w) = 12.3 +/- 0.3, compared with s(20,w) = 10.5 +/- 0.3 of the free e nzyme, indicating large changes in the hydrodynamic properties of the enzyme in the complex. These results provide direct evidence that the DnaB hexamer undergoes dramatic conformational changes which include a ll six subunits of the enzyme in the ternary complex. Moreover: sedime ntation velocity studies of the ternary complex provide direct evidenc e that the hexamer is the species which binds ss nucleic acid. The sig nificance of these results for a mechanistic model of the functioning of the DnaB helicase in DNA replication is discussed.