The Isolation and Preliminary Characterization of Endonoclease VIII from Escherichia Coli
Free radicals are highly reactive chemical species that are generated by a number of different mechanisms, including x-rays, certain chemical carcinogens and even aerobic metabolism. In order to survive the damages caused by free radical species, cells have evolved a number of protective mechanisms (5). Since DNA is an important target for free radicals, the ability to repair radical-induced DNA damages should constitute a significant cellular survival response. For a number of years our laboratory has been involved in identifying, quantitating, and assessing the biological impact of X-ray-induced DNA damages (26, 27), many of which are produced by free radicals (2,10). Since the 5–6 double bond of thymine is particularly susceptible to free radical attack (5), enzymatic, chemical and immunological methods have been used to focus our efforts on radical-induced oxidation products of thymine (26, 27). Endonuclease III from E. coli has been used as a tool to probe x-irradiated DNA for thymine ring saturation (15) and fragmentation (16) products. Furthermore, both thymine glycols and urea residues (a fragmentation product of thymine hydroperoxides) act as replicative blocks in vitro (4,9,12,25) and are lethal lesions in vivo (12,23).
KeywordsFluorometric Assay Fragmentation Product Free Radical Attack Methoxy Amine Replicative Block
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