The fluorescence quenching of the bacteriophage M13 encoded gene-5 protein was used to study its binding characteristics to different polynucleotides. Experiments were performed at different salt concentrations and in some instances at different temperatures. The affinity of the protein depends on the base and sugar composition of the polynucleotides involved and may differ appreciably, i.e. by orders of magnitude. The salt dependence of binding is within experimental accuracy equal for all single stranded polynucleotides. A method is presented to estimate values of the cooperativity constant from salt titration curves. These values are systematically higher than those obtained from titration experiments in which protein is added to a polynucleotide solution. A comparison is made between the binding constants of the gene-5 protein and the gene-32 protein encoded by the T4 phage. Possible implications of the binding characteristics of the gene-5 protein for an understanding of its role in vivo are discussed.

The binding of gene-5 protein, encoded by bacteriophage M 13, to oligodeoxynucleic acids was studied by means of fluorescence binding experiments, fluorescence depolarization measurements and irreversible dissociation kinetics of the protein . nucleotide complexes with salt. The binding properties thus obtained are compared with those of the binding to polynucleotides, especially at very low salt concentration. It appears that the binding to oligonucleotides is always characterized by a stoichiometry (n) of 2 -3 nucleotides/protein, and the absence of cooperativity. In contrast the protein can bind to polynucleotides in two different modes, one with a stoichiometry of n = 3 in the absence of salt and another with n = 4 at moderate salt concentrations. Both modes have a high intramode cooperativity (o about 500) but are non-interacting and mutually exclusive. For deoxynucleic acids with a chain length of 25 -30 residues a transition from oligonucleotide to polynucleotide binding is observed at increasing nucleotide/protein ratio in the solution. The n = 3 polynucleotide binding is very sensitive to the ionic strength and is only detectable at very low salt concentrations. The ionic strength dependency per nucleotide of the n = 4 binding is much less and is comparable with the salt dependency of the oligonucleotide binding. Furthermore it appears that the influence of the salt concentration on the oligonucleotide binding constant is to about the same degree determined by the effect of salt on the association and dissociation rate constants. Model calculations indicate that the fluorescence depolarization titration curves can only be explained by a model for oligonucleotide binding in which a protein dimer binds with its two dimer halves to the same strand. In addition it is only possible to explain the observed effect of the chain length of the oligonucleotide on both the apparent binding constant and the dissociation rate by assuming the existence of interactions between protein dimers bound to different strands. This results in the formation of a complex consisting of two nucleotide strands with protein in between and stabilized by the dimer-dimer interactions.It is now well-known, at least for prokaryotes, that singlestranded DNA-binding proteins participate in the process of DNA replication. The gene-5 protein encoded by the filamentous bacteriophage MI3 is a case in point. It binds strongly and cooperatively to single-stranded DNA and is able to destabilize double-stranded DNA. During DNA metabolism it is instrumental in the induction of a switch from replicativeform replication to single-strand DNA synthesis.Determinants in the DNA protein interaction are the stoichiometry and the nature of the binding. The question of the stoichiometry of the gene-5 protein/DNA binding, i. e. the number of nucleotides covered by one protein molecule, has been addressed in a number of studies [ 1 -61 and has been reviewed recently by Kansy et al. [7]. A so-called n = 4 binding mode, in which the protein molecule covers four ...

The binding of the bacteriophage-M13-encoded gene-5 protein to oligo(deoxythymidy1ic acid)s and MI 3 DNA was studied by means of tyrosyl fluorescence decay and fluorescence anisotropy measurements. The observed fluorescence decays could be described with two exponentials, characterised by the lifetimes T~ = 2.2 ns and z2 = 0.8 ns respectively. Only the amplitude of the longer-lifetime component is influenced by binding of the protein to DNA. This indicates that a part of the tyrosyl residues is involved in the binding. By means of fluorescence depolarisation measurements the rotational correlation time of the protein dimer is found to be 12.9 ns. In contrast to earlier measurements, carried out on the DNA- [85][86][87][88][89][90][91][92][93], the observed rotational correlation times of the gene-5 protein pass through a maximum when the protein is titrated with oligo(deoxythymidy1ic acid)s. This is not observed upon titration with M13 DNA. Our measurements showed that for the oligo(deoxythymidylic acid)s there clearly is a decrease in the number of clustered proteins on the lattice in the case of excess nucleotide. This is a direct consequence of the much lower cooperativity of the binding to the oligonucleotides compared to the cooperativity characteristic of binding to polynucleotides. The number of nucleotides covered by a protein monomer is found to be I 3 for the oligonucleotides and % 4 for M13 DNA. Model calculations show that the 'time-window' through which the fluorescence depolarisation can be observed (i.e. the fluorescence lifetime) in this case significantly affects the 'measured' effective rotational correlation times.An interesting aspect of the interaction between the singlestrand-DNA-binding gene-5 protein, encoded by the bacteriophage M13, is its ability to bind to DNA in two different modes, previously designated the oligonucleotide and the polynucleotide binding mode. In the polynucleotide binding mode the protein covers four nucleotides and the cooperativity of binding is high, i.e. the binding of a protein adjacent to an already bound protein is increased by a factor of five hundred (o = 500) with respect to binding to a naked lattice. In the so-called oligonucleotide binding mode the protein covers three nucleotides and the cooperativity of the binding is two orders of magnitude smaller (w w 5). Moreover, the salt dependence of the two binding modes differs significantly [l-31. Recently the binding characteristics of the single-strand-DNA-binding protein of the filamentous phage Pfl were studied in detail by means of fluorescence depolarisation measure- DNA. However, in contrast to the M13 gene-5 protein, for the Pfl protein different binding modes could not be distinghuished: complex formation to polynucleotides and oligonucleotides turned out to be very much the same. Thus the question arises whether the two proteins are intrinsically different in their DNA-binding properties. This is not self evident, because the second binding mode discovered for the M13 gene-5 protein was detected...

The irreversible dissociation kinetics of complexes of M13-encoded gene-5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped-flow experiments. A linear decay was found for all gene-5-protein . poly(dA) complexes and for the gene-5-protein . M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single-exponential decay was found for the dissociation of gene-5-protein . M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel [I] is not maintained.The binding of gene-5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA-binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide [2]. The absence of line broadening for gene-5-protein . polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.