Escherichia coli single-strand deoxyribonucleic acid binding protein: stability, specificity, and kinetics of complexes with oligonucleotides and deoxyribonucleic acid

Abstract

The complex formation between the single-strand DNA binding protein (ssB protein) from E. coli and oligonucleotides and single-stranded DNA was studied using fluorescence titrations, ultracentrifugation measurements and fast kinetic techniques. Determination of the stoichiometries of oligo(dT)-ssB complexes shows that each of the 4 subunits of the ssB protein represents a binding site for an oligonucleotide about 8 residues long. Occupation of all 4 binding sites with oligo(dT) or poly(dT) leads to 80% quenching of the intrinsic protein fluorescence. The binding sites are nearly equivalent and independent. For d(pT)16, the intrinsic binding constant is 6 .times. 105 M-1, and for d(pT)30-40, which is long enough to extend continuously over the ssB tetramer, the binding constant is higher than 5 .times. 108 M-1. Oligoadenylates bind about 2 orders of magnitude weaker than the corresponding oligo(dT) species. The binding of oligo(dT) is very weakly dependent on ionic strength, in contrast to the oligo(dA)-ssB complex formation. For d(pT)8, d(pT)16, and d(pT)30-40, the complex formation can be described by a simple 1-step reaction. The strength of the interaction is mainly expressed in the rate constant is dissociation. In the cooperative complexes with poly(dT) or poly(dA), all 4 binding sites on the ssB tetramer are also occupied. Single-stranded DNA is coiled around the ssB molecule. Fluorescence melting experiments of the complexes show that the conformation of the single-stranded DNA has a strong influence on the stability of the complexes.