Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

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Abstract
Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of .lambda. > 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, an endolytic activity was discovered in extracts of Micrococcus luteus that is directed towards 8-(2-hydroxy-2-propyl)purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or .lambda.-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA; however, .lambda.- or UV-(254 nm) irradiated double-stranded DNA do inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNA. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, the specific excision of the 8-alkylated purines from the damaged DNA could be demonstrated. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease.