MEASUREMENT OF DNA CROSSLINKS BY S1 NUCLEASE: INDUCTION AND REPAIR IN PSORALEN‐PLUS‐360 nm LIGHT TREATED ESCHERICHIA COLI

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Abstract
—DNA crosslinks in Escherichia coli cells. exposed to 4.5′,8‐trimethylpsoralen plus 360 nm light, were measured using a rapid and sensitive new approach. The assay is based on the specificity of S1 nuclease from Aspergillus oryzae to single‐stranded DNA. Bacterial cells were lysed and the DNA denatured by alkali. Following acid neutralization. crosslinked DNA undergoes spontaneous renaturation and is rendered S1‐nuclease resistant and therefore acid‐precipitable. The single‐stranded fraction after denaturation by alkali decreases with increasing near UV light exposure in the presence of TMP following first order kinetics. The kinetics were faster when exposure was at 4°C rather than at 20°C. This suggests that excision of crosslinks occurs during exposure at the higher temperature. Indeed. since the rate of DNA crosslinking in a uvr B mutant which is excision‐deficient was higher than in wild type bacteria at 4°C, some excision must have occurred even in the cold. DNA from excision‐proficient cells incubated at 37°C following exposure to TMP‐plus‐near UV at 4° showed a greater single stranded fraction than that from non‐incubated cells. This indicates repair of DNA crosslinks. which proceeded with a half‐time of 8 min at 37°C and was unaffected by substitution of thymine in DNA by 5‐bromouracil.

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