Benzamide-DNA interactions: Deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct

• Published in: Biochimica et biophysica acta Vol. 909; no. 1; pp. 71 - 83
• Main Authors: McLick, Jerome, Hakam, Alaeddin, Bauer, Pal I., Kun, Ernest, Zacharias, David E., Glusker, Jenny P.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 06.06.1987; Elsevier; North-Holland
• Subjects: Adenine - analogs & derivatives; Animals; Base Composition; Base Sequence; Benzamide; Benzamides - metabolism; Benzamides - pharmacology; Benzoates; Benzoic Acid; Biological and medical sciences; Carcinogenesis, carcinogens and anticarcinogens; Cattle; Cell Nucleus - metabolism; Chemical agents; DNA; DNA - metabolism; Kinetics; Liver - metabolism; Liver nucleus; Medical sciences; Models, Molecular; Molecular Conformation; Poly(ADP-ribose) Polymerases - metabolism; Poly(ADPribose) polymerase; Rats; Thymus Gland - enzymology; Tumors; X-Ray Diffraction; X-ray structure; Liver nucleus; Poly(ADPribose) polymerase; Benzamide; X-ray structure; DNA; Rat; Liver; Rodentia; Poly(ADP-ribose) polymerase; Molecular interaction; Cell transformation; In vitro; Site of action; Vertebrata; Mammalia; Animal; Mechanism of action; Molecular adduct; Crystalline structure
• ISSN: 0167-4781; 0006-3002; 1879-2634; 1878-2434
• DOI: 10.1016/0167-4781(87)90047-9

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.