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

Abstract

Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of X > 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 842-hydroxv-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, we discovered in extracts of Micrococcus luteus an endonucleolytic activity 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 y-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA; however, 'y-or UV-(254 nm) irradiated double-stranded DNAs do inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. 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 were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-a kylation 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. Nucleotide excision repair is one of the main repair mechanisms that acts to restore the structural integrity of damaged DNA. It is initiated by an endonuclease that incises the damaged DNA strand in the neighborhood of the damage, followved by excision of the damaged sequences by an exonuclease, repolymerization by a DNA polymerase, and finally covalent sealing by DNA ligase to restore the DNA molecule (1-3).The nucleotide excision repair is initiated by specific endonucleases that recognize chemically modified regions, or consequent conformational changes in the DNA, and incise the DNA at the vicinity of such lesions. So far only two endonucleases that are directed towards known chemically defined lesions have been described. These are the pyrimidine-dimer endonuclease (4-6) and the apurinic/apyrimidinic site endonuclease (7-13). Other endonuicleases have been found to incise UV-, 'y-ray-, or chemically damaged DNA, but their specificities are not yet known (14)(15)(16)(17)(18)(19)(20)(21)(22). This is mainly due to the fact that the damaged DNA substrates usually contain a multiplicity of products, not all of them chemically characterized, thus preventing an unambiguous assignment of a specific enzyme to a defined lesion. This difficulty interferes with the characterization of repair endontucleases and with the elucidation of the molecular basis of the damage to enzyme interactions, which play a key role in the initiation of the repair process.We have a...

Perturbations of enzymic uracil excision due to purine damage in DNA.

“…Therefore, the major products ofphotoalkylation ofPBS-2 DNA were photoalkylated purines. As with native thymine-containing DNAs, no photoalkylated pyrimidines were detected (9,14). This is consistent with the suppression of photoalkylation of pyrimidines by equimolar purines, reported for both uracil and thymine bases (28) and for PM2 DNA (9).…”

“…HPG marker was made by photoalkylation of guanine (16). Identities of both were confirmed by paper chromatography (9,14). mGua was obtained from Vega Biochemicals (Tucson, AZ).…”

“…The rate of enzymic release of uracil was markedly decreased when the The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 (9), except that the samples were not flushed with nitrogen before irradiation. Actinometry (10) showed the incident dose to be 6.6 x 10-5 einstein cm-2 min-1.…”

Purine Photoproducts*

“…Covalently-bound DNA purine-carcinogen adducts have been isolated from the reaction products, but destruction of purines has also been reported (Strniste et al, 1980). It has not been determined if the sDectrum of Droducts depurination by heating the DNkS in acid, the bases were separated by paper chromatography and the ratios determined according to Livneh et al (1979). Each point represents the average of two determinations.…”

“…An endonuclease activity was detected in crude extracts of M . luteus that incised photoalkylated DNA (Livneh et al, 1979). This activity was neither purified nor characterized.…”