Accelerated Publications Negative Cooperativity within Individual Tetramers of Escherichia coli Single Strand Binding Protein Is Responsible for the Transition between the (SSB)35

W e have examined the binding of the oligonucleotide ~ T ( P T ) ~ ~ to the Escherichia coli SSB protein as a function of NaCl and MgC12 concentration (25 OC, pH 8.1) by monitoring the quenching of the intrinsic protein fluorescence. We find two binding sites for ~ T ( P T ) ~ ~ per single strand binding (SSB) protein tetramer, with each site possessing widely different affinities depending on the salt concentration. At 200 m M NaCl, we observe nearly stoichiometric binding of dT(pT),, to both binding sites within the SSB tetramer, although a difference in the affinities is still apparent. However, when the NaCl concentration is lowered, the overall affinity of ~ T ( P T ) ~ ~ for the second site on the SSB tetramer decreases dramatically. At 1.5 m M NaCI, only a single molecule of ~ T ( P T ) ~ ~ can bind per SSB tetramer, even with a 10-fold molar excess of ~ T ( P T ) ~ ~ . MgC12 is effective at 100-fold lower concentrations than NaCl in promoting the binding of the second molecule of ~ T ( P T ) ~ ~ . This binding behavior reflects an intrinsic property of the SSB tetramer, since it is also observed upon binding of smaller oligonucleotides, and the simplest explanation is that a salt-dependent negative cooperativity exists between D N A binding sites within the SSB tetramer. This phenomenon is also responsible for the transition between the two SSB-single strand (ss) polynucleotide binding modes that cover 35 and 56 nucleotides per tetramer [Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-78021, Extreme negative cooperativity stabilizes the (SSB)35 binding mode, in which the SSB tetramer binds tightly to ss DNA with only two of its subunits while the other two subunits remain unligated. At higher salt concentrations, negative cooperativity is reduced with the result that all four SSB subunits can interact with ss DNA, as in the (SSB)56 and (SSB),, binding modes. The possible biological significance of this negative cooperativity is discussed. %e Escherichia coli single strand binding (SSB) protein is a helix-destabilizing protein that is required for DNA replication and a variety of repair processes in that organism (Sigal et al., 1972; Chase & Williams, 1986). It also stimulates the DNA strand exchange activity of the recA protein and hence is important in homologous recombination (Cox & Lehman, 1987). In these respects it is similar to the bacteriophage T4 gene 32 protein, although the E. coli SSB protein differs greatly in structural detail, as well as in its interactions with single-stranded (ss) nucleic acids. The SSB protein is a stable tetramer possessing D2 symmetry (Ollis et al., 1983), and its tetrameric structure is maintained upon binding oligonucleotides (Bandyopadhyay & Wu, 1978; Krauss et al., 1981). The protein binds selectively and cooperatively to ss polynucleotides (Sigal et al., 1972; Lohman et al., 1986a; Bujalowski & Lohman, 1987a), although the interactions are T.M.L. is a recipient of American Cancer Society Faculty Research Award FRA-303. This work was supported in part by NIH Grant GM-30498 and Robert A. Welch Foundation Grant A-898 (to T.M.L.) and by NIH Biomedical Research Support Instrumentation Grant SO1 RR01712 and DOD Instrumentation Grant P-20862-LS-RI. Support from the Texas Agricultural Experiment Station is also acknowledged. * Address correspondence to this author at the Department of Biochemistry and Biophysics. *Department of Biochemistry and Biophysics. "On leave from the Institute of Biology, Department of Biopolymer Department of Chemistry. Biochemistry, Poznan University, 61-701 Poznan, Poland. 0006-2960188 10427-2260$01.50/0 complex since multiple binding modes can form on ss polynucleotides, depending on the solution conditions, particularly the salt concentration and type (Lohman & Overman, 1985; Bujalowski & Lohman, 1986; Griffith et al., 1984; Chrysogelos & Griffith, 1982). In studies with poly(dT), several binding modes have been identified for the E. coli SSB protein, and the transitions among the binding modes are dependent on cation and anion type and concentration, pH, temperature, and protein binding density (Lohman & Overman, 1985; Bujalowski & Lohman, 1986; Bujalowski et al., 1988). Different binding density dependent morphologies of SSB protein-ss M13 DNA complexes have also been observed by electron microscopy (Griffith et al., 1984). At 25 OC, pH 8.1, three major binding modes that differ in the number of nucleotides occluded by the SSB tetramer (Le., the site size n) have been observed with n = 35, 56, and 65 nucleotides per SSB tetramer (Lohman & Overman, 1985; Bujalowski & Lohman, 1986). In the absence of dior polyvalent cations, the (SSB),, mode is stable below 10 mM NaCl at high protein binding densities; the (SSB)56 mode is stable in the region from 50 mM I [NaCl] I 0.1 M, whereas above 0.2 M NaC1, the (SSB),, mode is stable (Bujalowski & Lohman, 1986). MgCI, is much more effective than NaCl in promoting the transition from the (SSB)35 to the (SSB)56 binding mode, such that the (SSB),, mode is stable in the range from 4 to 50 mM MgCl,. It has been proposed that, in the (SSB),, complex, the ss DNA interacts with only