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Abstract— Following UV irradiation at 254 nm and treatment with hot piperidine, single‐stranded 49‐mer and 12‐mer oligodeoxyribonucleotides have been shown by gel‐sequencing experiments to contain a prominent alkali‐labile cleavage site that maps to adenine in the sequence element 5′‐TTGATC‐3′. This behavior is abolished by single base substitutions within the photoreactive tract and does not occur with duplex DNA. The distinctive properties of the photolesion are consistent with the formation of an abasic site through initial loss of a photomodified adenine base of unknown structure. The presence of an abasic site is supported by the observations that the alkaline cleavage fragments are terminally phosphorylated and that strand scission can also be effected by spermidine and the tripeptide Lys‐Trp‐Lys.

Far-UV irradiation induces photoaddition between adjacent thymine and adenine bases on the same strand of DNA to give the mixed purine-pyrimidine photoadduct, TA* [ 1,2]. Conveniently, treatment with acid specifically converts TA* into the highly fluorescent, heterocyclic base, 6-methylimidazo [4,. whose formation is diagnostic of the parent photoproduct [3]. Although 6-MIP may be isolated and quantified by paper electrophoresis and fluorimetry, respectively, radiochemical detection coupled to HPLC affords greater sensitivity and is therefore the preferred approach when diminished TA* yields are anticipated. Since the methyl group of the thymine moiety is retained in 6-MIP, estimation of TA* is possible by scintillation counting of the 6-MIP isolated by HPLC from acid hydrolysates of DNA labelled with [rnefhyl-3Hlthymidine. In this way, the quantum efficiency of TA* formation has been calculated to be -1 x 10-5 mol einstein-I in naked DNA [4], implying that it is a relatively rare photolesion. The more abundant types of DNA photolesion such as pyrimidine dimers and pyrimidine (6-4) pyrimidone photoadducts have been widely detected and quantified in UV-irradiated cells [5]. In contrast, no attempts have been made hitherto, to search for and quantify the TA* photoproduct in the DNA of UV-irradiated intact cells. By using an adapted version of the radiochemical assay devised by Bose and Davies [4], we report here that TA* is formed in a cellular environment.Cultured human epithelial (HEp-2) cells were radiolabelled to high specific activity with [rnethyl-3H]thymidine. This was accomplished by initially inducing partial cell synchrony using repeated subculture, followed by arresting the cell cycle at the GUS-phase boundary using aminopterin [6], and then providing a Irneihyl-3Hlthymidine pulse. Cell irradiation was carried out using two 8 W germicidal strip lamps emitting predominantly at 254 nm and mounted in parallel. The cell monolayers, under phosphate buffered saline at 4 W , were positioned 8 cm below the UV source and irradiated with an incident UV fluence of 10 kJ m-2 (measured by actinometry with 1,3-dimethyluracil [7]). The radiolabelled, UV-irradiated DNA was then extracted, recovered by ethanol precipitation and hydrolysed with 1 M HCI at IOOOC for 4 h. The resulting hydrolysate was lyophilised, redissolved in 0.05% TFA and spiked with a known amount of unlabelled synthetic 6-MIP. This permitted the location of 6-MIP formed from photogenerated TA* during subsequent purification steps. The spiked hydrolysates were then subjected to analytical reversed-phase HPLC using a Techopak C 18 column (30 0 x 3.9 mm, from HPLC Technology Ltd.) Following sample injection, the column was eluted isocratically with 0.05% TFA for 5 min, before applying a linear gradient to 30% methanol in 0.05% TFA over 5 min. This solvent mix was maintained for 10 min, followed by a linear return to starting conditions. The flow rate was 0.8 mi min-1 and the elution profile was monitored at 254 nm. 6-MIP fractions collected from multiple...