Dissociation of the Pf1 nucleoprotein assembly complex and characterisation of the DNA binding protein.
During replication of bacteriophage Pf1, progeny viral strands are complexed with a single-stranded DNA binding protein, analogous to the gene 5 protein of bacteriophage fd. Using fluorescence spectroscopy, ultracentrifugation and DNA-cellulose chromatography, conditions for dissociation of the nucleoprotein have been investigated. The Pf1 protein is unusual in that it is not released from the DNA by 2 M NaCl. Complete separation occurs in 0.6-1.0 M MgCl2, leading to a procedure for the purification of the protein. Two subfractions of the protein can be isolated of isoelectric points 5.9 and 6.4. The molecular weight of the native DNA binding protein has been studied by gel filtration and sedimentation. The major species in solution has a sedimentation coefficient of 2.3 S and a diffusion coefficient of 7.8 X 10(-7) cm2 . s-1, corresponding to a protein dimer (Mr = 30 800). Protein tetramers are induced in the presence of octanucleotides, but not tetranucleotides. Analysis of the ultraviolet spectra of the DNA binding protein and the native nucleoprotein complex indicates a stoichiometry of 3.9 +/- 0.4 nucleotides per protein subunit. The molar extinction coefficient of the DNA when bound to the protein (epsilon 260 = 8100) suggests that the binding protein maintains the DNA in an extended (unstacked) conformation similar to that found in the mature Pf1 virion.
D001435:Bacteriophages / Q000235:genetics*; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000302:isolation & purification*; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins / Q000302:isolation & purification*; D008274:Magnesium; D009698:Nucleoproteins / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D012964:Sodium; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D009698:Nucleoproteins; D012964:Sodium; D004265:DNA Helicases; D008274:Magnesium