DNA‐binding properties of gene‐5 protein encoded by bacteriophage M 13

Abstract

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.

Role of Tyr-22 in the Binding of Pf3 ssDNA Binding Protein to Nucleic Acids

“…The CD and mobility shift experiments are consistent with two exclusive modes of binding for the Pf3 ssDBP wildtype and Y22F proteins, as previously has been shown to be the case for the fd g5p (Kansy et al, 1986;Bulsink et al, 1988). That is, the binding may be qualitatively described as being consistent with two modes having large ments (Kansy et al, 1986).…”

“…In this illustration, the CD change per nucleotide bound in the n = 4 mode is 0.7 that of a nucleotide bound in the n = 2 mode. It should be noted that a maximum CD effect at P/N s 0.25 by binding in an n = 4 mode does not require that the DNA lattice be saturated at this P/N ratio (Kansy et al, 1986;Bulsink et al, 1988). [nucleotide] increased above 0.25, the slopes of the titration plots with the mutant protein decreased, to give total CD changes at saturation that were qualitatively the same as for titrations with the wild-type protein.…”

Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR

“…As the number of RF DNA molecules increases, so does the concentration of the phage proteins present in the host cell. When gVp reaches a threshold concentration in the host cell, it binds cooperatively to a hairpin on the ssDNA and coats its entire length with approximately 1500 proteins per ssDNA, thereby forming an intermediate complex that prevents conversion into RF DNA [9]. gVp is essential for progeny ssDNA production [10].…”

Crystal Structures of Y41H and Y41F Mutants of Gene V Protein from Ff Phage Suggest Possible Protein-Protein Interactions in the GVP-ssDNA Complex

“…DNA binding is triggered by the cellular GVP concentration in the host Escherichia coli. Substantial biochemical and biophysical data have been accumulated regarding the interactions between GVP and DNA (Fulford & Model, 1988;Gray, 1989;Model et al, 1982;Zaman et al, 1992;Anderson et al, 1975;Dick et al, 1988;Bulsink et al, 1986Bulsink et al, , 1988Kansy et al, 1986). NMR and fluorescence studies have suggested that certain amino acids of GVP are in contact with DNA in the GVP-ssDNA complex (Dick etal., 1988;Stassenetal., 1992a,b).…”