Livneh Z, Elad D, Sperling J
Proc Natl Acad Sci U S A. 1979 Nov;76(11):5500-4. doi: 10.1073/pnas.76.11.5500.
Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 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, 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 gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to 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-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.
使用自由基光引发剂和波长大于305nm的紫外光，噬菌体PM2的环状共价闭合DNA与异丙醇发生光烷基化反应，导致DNA中嘌呤部分发生特异性的8-取代，产生8-(2-羟基-2-丙基)腺嘌呤和8-(2-羟基-2-丙基)鸟嘌呤，这是DNA中唯一可检测到的损伤。以这种经过特异性光烷基化的DNA为底物，我们在藤黄微球菌提取物中发现了一种内切核酸酶活性，该活性针对DNA中的8-(2-羟基-2-丙基)嘌呤。该活性不是DNA糖基化酶和无嘌呤位点内切核酸酶的组合。它不受单链DNA、紫外线或γ射线照射的单链DNA，或正常或脱嘌呤双链DNA的抑制。然而，γ射线或紫外线(254nm)照射的双链DNA会抑制该活性，这暗示了这些受损DNA中可能存在一种常见类型的损伤。切割活性不需要二价阳离子，在1mM EDTA中它完全有活性，而咖啡因和ATP会导致抑制。发现缺乏嘧啶二聚体导向内切核酸酶的藤黄微球菌突变体提取物中内切核酸酶活性水平与野生型相当。切割后，我们可以证明从受损DNA中特异性切除8-烷基化嘌呤。嘌呤8-烷基化在核苷酸水平上的特殊构象后果，即它们不规则的顺式构象，表明内切核酸酶识别的是DNA中的扭曲。
