PubMed1991
A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.