Selective alkylation of carcinogenic 9-anthryloxirane at the N-3 position of adenine in DNA.

Abstract

Carcinogenic 9-anthryloxirane binds covalently to calf thymus DNA and poly(dA-dT). Application of the technique for DNA sequence determination shows that acid cleavage of the modified DNA frees approximately half of the anthryl groups from the DNA. HPLC analysis indicates that an adenine adduct and the glycol derived from 9-anthryloxirane are the major acid-labile products. Spectroscopic analyses establish that the adenine adduct is the N-3 adduct of 9-anthryloxirane to adenine. Similar analyses of modified poly(dA-dT) indicate that the binding of 9-anthryloxirane takes place selectively at the N-3 position of adenine. The significance of this finding is briefly discussed. MATERIALS AND METHODSMaterials. Highly polymerized calf thymus DNA, hyperchromicity 30%, was purchased from Sigma, and poly(dAdT) was from the Pharmacia P-L Biochemicals. Solvents and reagents used were of the highest available purity commercially. 9-AO was synthesized by a known method (6).Instruments. All UV-visible absorption spectra were taken on a Varian-Cary 219 spectrometer; NMR spectra, on a University of Chicago DS-1000 spectrometer operating at 500 MHz; and mass spectra, on a VG-70-250 double-focusing integrated GS-MS-GS mass spectrometer. All HPLC were performed on a custom system from Waters Associates with a RCM-100 radial compression module.Reaction of 9-AO with Calf Thymus DNA. A solution of calf thymus DNA (10 mg) in 10 ml of 5 mM sodium cacodylate buffer (pH 7.1) was divided into two equal 5-ml portions. One portion was denatured by heating at 95°C for 5 min and quenched in an ice-water bath for 10 min. Both portions were then treated with a solution of 5 mg of 9-AO dissolved in 0.25 ml of (CH3)2SO, and the reaction mixtures were incubated at 37°C in the dark for 24 hr. Each solution was extracted 14 times with 5 ml of buffer-saturated ethyl acetate and twice with 5 ml of buffer-saturated ether to remove organic compounds that were not covalently bound to DNA. The solutions were then evaporated at 40 torr (1 torr = 133.3 Pa) and 21°C to remove the dissolved organic solvents and were made up to 5 ml (total volume) with distilled water. Absorption spectra ofthe solutions were then recorded. The binding level of I to DNA was estimated from the absorbance at 385 nm, and the value is given in Table 1. A portion of the modified native DNA was heat-denatured as described, and the resulting solution was extracted with buffer-saturated ethyl acetate (six times) and ether (twice). Absorption spectra of the modified native DNA showed no significant decrease in absorption before and after such a treatment.Each portion of the modified DNA was then hydrolyzed by acidifying 3 ml of the solution with 0.1 ml of 1 M HC1 to pH 1.5 and heating the solution at 60°C for 1 hr. Acid-labile products were removed by extraction with six 3-ml portions of buffer-saturated dichloromethane. The dichloromethane extracts were combined, dried, and evaporated. The residue was dissolved in 1.5 ml of CH30H. Absorption spectra of the Abbreviations: 9-AO, ...