Four distinct binding modes for the interaction of Escherichia coli single-strand binding (SSB) protein with single-stranded (ss) DNA have been identified on the basis of quantitative titrations that monitor the quenching of the SSB protein fluorescence upon binding to the homopolynucleotide poly(dT) over a range of MgCl2 and NaCl concentrations at 25 and 37 °C. This is the first observation of multiple binding modes for a single protein binding to DNA. These results extend previous studies performed in NaCl (25 °C, pH 8.1), in which two distinct SSB-ss DNA binding modes possessing site sizes of 33 and 65 nucleotides per bound SSB tetramer were observed [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603]. Each of these binding modes differs in the number of nucleotides occluded upon interaction with ss DNA (i.e., site size). Along with the previously observed modes with site sizes of 35 ± 2 and 65 ± 3 nucleotides per tetramer, a third distinct binding mode, at 25 °C, has been identified, possessing a site size of 56 ± 3 nucleotides per bound SSB tetramer, which is stable over a wide range of MgCl2 concentrations. At 37 °C, a fourth binding mode is observed, possessing a site size of 40 ± 2 nucleotides per tetramer, although this mode is observable only over a small range of salt concentration. The relative populations of each binding mode are modulated primarily by the charge and concentration of low molecular weight cations in solution (Mg2+ and Na+ in these experiments), indicating that a net binding of cations occurs upon formation of each of the higher site size SSB—DNA complexes. Mg2+ is much more effective than Na+ in facilitating the transitions to the higher site size binding modes. At 25 °C (pH 8.1) the two binding mode transitions have midpoints of 0.6 and 64 mM in MgCl2 and 17 mM and 0.16 M in NaCl; hence, all three SSB binding modes may form in vivo since these salt concentrations are within the range estimated to occur in E. coli. These transitions also occur within the same range of MgCl2 concentrations used for replication and recombination studies in vitro; hence, they are of definite importance for any such studies in vitro. Since the E. coli SSB protein is essential for replication, recombination, and repair processes, it is possible that the different binding modes identified here are used selectively in each of these processes in vivo.
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