DNA "melting" proteins. I. Effects of bovine pancreatic ribonuclease binding on the conformation and stability of DNA.
Bovine pancreatic ribonuclease is a DNA "melting" protein, since it binds with greater overall affinity to the single-stranded than to the double-stranded form of natural and synthetic deoxyribose-containing polynucleotides. As such, the DNA-RNase system provides a simple model for the more complex and biologically relevant melting protein-nucleic acid systems. Aspects of the DNA-RNase interactions which are related to the quantitative assessment of this system as a melting protein model are investigated here. A boundary sedimentation velocity technique is used to measure thermodynamic parameters of the interaction; association constants (Kh and Kc) and site sizes (nh and nc) are determined for the interaction of ribonuclease with native (double helical) and denatured (random coil) DNA. It is shown that log Kh and log Kc are linear functions of log [Na+], binding decreasing with increasing Na+ concentration, with Kh about 2 orders of magnitude smaller than Kc at the ionic strengths studied, nh and nc are approximately 8 and approximately 11 nucleotide residues, respectively, indicating that potential binding sites overlap. Binding to both forms of DNA is non-cooperative. It is shown by CD and ultraviolet spectroscopy that the binding of RNase to single- and double-stranded DNA perturbs the conformations of these polynucleotide conformations very little relative to the unliganded structures. Hydrodynamic methods are used to show that RNase binds to native DNA without altering the overall solution structure of the latter; however conditons which permit binding to, and stabilization of, transiently exposed single-stranded sequences result in a collapse of the stiff native DNA structure. We demonstrate by melting transition studies that ribonuclease does bring about an equilibrium destabilization of native DNA and poly [d(A-T)] and, by applying a ligand-perturbed helic in equilibrium coil theory developed by McGhee (McGhee, J.D. (1976) Biopolymers 15, 1345-1375), it is shown that the extent of the observed destabilization is in semiquantitative accord with expectations based on the measured affinity constants and site sizes for RNase binding to both DNA conformations. Spectral methods are used to show that the relative stability of native DNA sequences of varying base composition is the same in the presence and absence of ribonuclease, strongly arguing that this "melting" ligand "traps" single-stranded sequences transiently exposed by thermal fluctuations. RNase also undergoes an order in equilibrium disorder conformational transition as a function of temperature (the denatured form of RNase stabilizes native DNA, while native RNase destabilizes the native double helix), and the coupled equilibria involved in these interacting conformational changes are interpreted and discussed as possible models of genome regulatory interactions.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D003090:Coliphages; D004247:DNA; D004279:DNA, Viral; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009994:Osmolar Concentration; D010179:Pancreas / Q000201:enzymology; D011089:Polydeoxyribonucleotides; D012260:Ribonucleases; D012964:Sodium; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013950:Thymus Gland
D004279:DNA, Viral; D011089:Polydeoxyribonucleotides; D004247:DNA; D012964:Sodium; D012260:Ribonucleases