Interaction of a DNA-binding protein, the gene product of D5 of bacteriophage T5, with double-stranded DNA. Analysis by metrizamide gradient centrifugation.
Interactions of DNA and the gene product D5 (gpD5) of bacteriophage T5, a DNA-binding protein that binds preferentially and cooperatively to double-stranded DNA, were analyzed by metrizamide gradient centrifugation. Conditions were set so that DNA and DNA protein complex sedimented to apparent equilibrium positions. DNA has a buoyant density of 1.12 g/cm3, and DNA saturated with gpD5 has a buoyant density of 1.17 g/cm3. These values are independent of DNA size and base composition in the range studied. At gpD5 concentration below the saturation value in a low ionic strength buffer, DNA distribution is bimodal, indicating cooperative binding of gpD5 to DNA. However, in the presence of 10 mM MgCl2, the binding process becomes distributive, with the buoyant density increasing linearly with the amount of gpD5 added until the saturation. From these data, one molecule of gpD5 is calculated to cover 40 base pairs at saturation. The technique as described has general applicability to the study of any interaction between DNA and dNA-binding proteins that bind in sufficient amount to cause detectable changes in buoyant density.
D002499:Centrifugation, Density Gradient; D004265:DNA Helicases / Q000235:genetics* / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D008793:Metrizamide; D013604:T-Phages / Q000235:genetics*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004265:DNA Helicases; D008793:Metrizamide