The detection and characterization of rat protein recognizing DNA damaged by N-acetoxy-acetylaminofluorene
Proteins recognizing DNA damaged by the chemical carcinogen N- acetoxy-acetylaminofluorene (AAAF) were analyzed in nuclear extracts from rat tissues, using a 36 bp oligonucleotide as the substrate and an electrophoretic mobility shift assay. Two major proteins that form complexes with DNA damaged by AAAF were detected; one of them also bound DNA damaged by cis-diammine-dichloroplatinum. The complex specific for AAAF-damaged DNA contained protein loosely attached to nuclear components. It was extracted with 0.1 M NaCl. The amount of this protein was estimated at about 105 copies per liver cell nucleus, and its probable size was about 42 kDa as detected by the Southwestern blotting assay. Its affinity for DNA damaged by AAAF was ~10-fold higher than that for undamaged DNA. Analogous AAAF- DDB (damaged-DNA-binding) proteins were also detected in extracts from rat brain, testis and kidney tissue. The levels of such proteins were not affected in rats treated with the carcinogen 2-acetylaminofluorene.
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