A rapid and sensitive assay for pyrimidine dimers in DNA

We have developed a rapid, sensitive assay for pyrimidine dimers. The assay has greatly facilitated the purification and characterization of the photoreactivating enzyme. The procedure depends on (1) the resistance of the nucleotide phosphate bond in dimer-containing regions of DNA to attack by DNase I, venom phosphodiesterase and alkaline phosphatase and (2) selective adsorption to Norit of mononucleosides and ³²P-labeled, dimer containing oligonucleotides (but not ³²P₁) resulting from nuclease digestion of highly-purified, ³²P-labeled bacteriophage DNA. The method is sensitive and rapid. The presence of the usual nuclease activities found in cell extracts does not interfere with the assay. Thus photoreactivating enzyme activity can be detected even in the presence of non-specific or uv-specific nucleases. Neither photoreactivation nor the digestion reaction is affected by purification agents at concentrations commonly used in enzyme purification.