DNA‐binding properties of gene‐5 protein encoded by bacteriophage M13

Abstract

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 ...

Electrostatic potential distribution of the gene V protein from Ff phage facilitates cooperative DNA binding: A model of the GVP‐ssDNA complex

“…8, 10). This could explain the high cooperativity of the GVP binding to DNA (w > 500) (Bulsink et al, 1988a). Our model may also help explain the n = 3 binding mode for the oligonucleotide binding by GVP.…”

“…We wanted to show that those channels indeed have the suitable size and shape to accommodate the proper length of an ssDNA. In the active GVP-ssDNA complex found in the M 13 phage-infected E. coli, there are four nucleotides bound per protein monomer (Bulsink et al, 1988b). We inspected the environment of the channel and noted that the repeating distance between two equivalent positions of the adjacent dimers is -17 A.…”

“…Other studies indicated that two antiparallel ssDNA strands occupy the interior of the helix (Gray et al, 1982) and GVP binds to polymeric DNA and oligonucleotide, respectively, at a ratio of four and three nucleotides per protein monomer (Bulsink et al, 1988b). More detailed studies regarding the interactions between GVP and DNA have been carried out (reviewed in Kansy et al, 1986).…”

Structure of the gene V protein of bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl protein.

“…This loop includes residues [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] (Fig. 2 6 Harker peak height is the ratio of the value of the Patterson function at the lowest expected self-vector to the rms of the origin-removed map.…”

“…Gene V protein binding to single-stranded nucleic acids and to oligonucleotides has been studied using chemical modification, spectroscopic techniques, and mutagenesis (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The structure of the protein-ssDNA complex has been studied using electron microscopy and solution scattering methods and is found to consist of a regular left-handed superhelix in which the gene V protein dimers are arrayed on the outside of the superhelix and the ssDNA strands are inside (8,25).…”