Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

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Abstract
Brain chromosomal DNA isolated from fetal BDIX‐rats 1 h after i.v. administration of the ethylating N‐nitroso carcinogen N‐ethyl‐N‐nitrosourea (75 micrograms/g body weight), statistically contained one molecule of O6‐ethyl‐2′‐deoxyguanosine (O6‐EtdGuo) per 81 micron of DNA, as determined in enzymatic DNA hydrolysates by competitive radio‐immunoassay using a high‐affinity anti‐(O6‐EtdGuo) monoclonal antibody (ER‐6). After fragmentation of the DNA by the restriction enzyme AluI (average fragment length, Lav = 0.28 micron = 970 bp; length range, Lr = 1.87‐0.02 micron = 6540 ‐ 60 bp), a small (approximately 2%) fraction of DNA enriched in specific polypeptides tightly associated with DNA was separated from the bulk DNA by a glass fiber binding technique. As analyzed by immune electron microscopy, approximately 1% of the DNA molecules in this fraction contained clusters of 2‐10 (O6‐EtdGuo)‐antibody binding sites (ABS). On the cluster‐bearing fragments (Lav, 0.85 micron +/‐ 0.50 micron S.D.; corresponding to 2970 +/‐ 1760 bp) the average ABS‐ABS interspace distance was 110 nm (= 390 bp; range approximately 9‐600 nm), indicating a highly non‐random distribution of O6‐EtdGuo in target cell DNA.