Interaction of Lambda cro Repressor with Synthetic Operator 0_R3 Studied by Competition Binding with Minor Groove Binders

Abstract

In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F.

Mixed Mode of Ligand-DNA Binding Results in S-Shaped Binding Curves

“…In many studies of ligand binding with nucleic acids, both experimental and theoretical, S-shaped curves were obtained (13)(14)(15)(16)(17)(18)(19). However, in all these cases (16)(17)(18)(19), such curves appeared either when positive cooperative interactions were introduced into the model, or in the case of competitive binding of different ligands to DNA. In this paper we provide evidence that the S-shaped ligand-DNA binding curves may appear in the absence of the positive cooperative interaction between the bound ligand molecules, as a result of the "mixed mode" of DNA binding by the same ligand molecule.…”

“…Some of the anticancer agents are known to exhibit higher anticancer activity in the presence of cooperative interactions between them whereas other are more active in the "monomeric" form (11,12). In many studies of ligand binding with nucleic acids, both experimental and theoretical, S-shaped curves were obtained (13)(14)(15)(16)(17)(18)(19). However, in all these cases (16)(17)(18)(19), such curves appeared either when positive cooperative interactions were introduced into the model, or in the case of competitive binding of different ligands to DNA.…”