Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct.
The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.
D000225:Adenine / Q000031:analogs & derivatives*; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001549:Benzamides / Q000378:metabolism* / Q000494:pharmacology; D001565:Benzoates; D019817:Benzoic Acid; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D051381:Rats; D013950:Thymus Gland / Q000201:enzymology; D014961:X-Ray Diffraction
D001549:Benzamides; D001565:Benzoates; C001172:9-ethyladenine; C037689:benzamide; D019817:Benzoic Acid; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D000225:Adenine