Effects of minor and major groove-binding drugs and intercalators on the DNA association of minor groove-binding proteins RecA and deoxyribonuclease I detected by flow linear dichroism.
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 I (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'-O-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 interactions.
Tuite
E
E
Department of Physical Chemistry, Chalmers University of Technology, Göteborg, Sweden.
Sehlstedt
U
U
Hagmar
P
P
Nordén
B
B
Takahashi
M
M
D002942:Circular Dichroism; D004247:DNA / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004996:Ethidium / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry*; D008024:Ligands; D008739:Methyl Green / Q000737:chemistry; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011938:Rec A Recombinases / Q000737:chemistry*; D013057:Spectrum Analysis; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D007211:Indoles; D007364:Intercalating Agents; D008024:Ligands; C007293:DAPI; D008739:Methyl Green; D004247:DNA; D011938:Rec A Recombinases; D003850:Deoxyribonuclease I; D004996:Ethidium