The binding of the intercalating dye methylene blue (MB) to chromatin and to free DNA has been studied as a function of ionic strength at very low binding ratios (1 MB/400 DNA bases) using absorption spectroscopy. With increasing salt concentration MB is displaced from chromatin to a higher extent than from DNA. The free energy change for MB binding to chromatin is found to be approximately 5 kJ/mole lower than for binding to DNA. This difference can be explained by the reduced number of high affinity binding sites in chromatin due to the presence of histone proteins. The difference in binding energy is virtually independent of the degree of chromatin condensation and also of the valence of counter ions, suggesting that neither the affinity for, nor the number of intercalation sites in the linker DNA is markedly changed upon the salt-induced condensation. The unaffected thermodynamics of the linker binding suggests that factors such as DNA superhelicity and the electrostatic influence from the chromatosomes remain unchanged during chromatin condensation.
Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz. RecA protein from Escherichia coli and deoxyribonuclease 1 (bovine pancreas). Ligands representing three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators). Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double-strand cleavage by deoxyribonuclease I. Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove. Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non-hydrolyzable cofactor adenosine 5'-0-3-thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy. Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA-protein intcractions.
The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids. It is a homodimer of 57 600 subunit molecular weight and has a binding site for ATP and weak ATPase activity. In the presence of ATP, TyrR binds tyrosine, which induces self-association of TyrR from a dimer to a hexamer. This report examines the interaction of TyrR with a 42 bp DNA oligonucleotide containing a centrally located binding site for TyrR (TyrR box). Replacement of a thymidine residue with an aminouridine residue at positions 7, 9, 13, 15, 19, 22, and 26 from one end of the 42mer enables labeling with fluorescein and successive placement of the label along the major groove of the DNA. The fluorescence footprinting of the oligonucleotide was followed using steady-state and time-resolved fluorescence methods. Binding of the TyrR dimer caused significant changes in the fluorescent properties of the labels attached to positions 13, 15, and 26, suggesting the involvement of these bases in the binding of the protein. Except for the position 15 conjugate, binding of the TyrR dimer caused little change in fluorescence intensity. Therefore, fluorescence anisotropy was used to follow the binding equilibrium. The fluorescence of the position 15 conjugate increased 1.6-fold on binding TyrR, suggesting that the fluorophore was in close contact with the protein. For all conjugates, the addition of tyrosine at the end of the titration with TyrR increased the anisotropy markedly, suggesting that the hexameric form of TyrR could bind the oligonucleotide. Two rotational correlation times were found for the labeled conjugates: one reflecting the motion of the probe at its point of attachment to the DNA (220-290 ps), the other reflecting the global tumbling of the labeled oligonucleotide (14-21 ns). On binding TyrR, changes in the correlation times and their associated amplitudes and changes in the range of angular motion of the probe depended on the position of the label. Evidence is presented that the binding of the TyrR hexamer, but not the TyrR dimer, affects regions that flank the binding sequence. The results support the hypothesis that the binding of the TyrR hexamer is responsible for interaction between tandem TyrR boxes in the tyrR regulon.
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