High-pressure liquid chromatography assay for quantitatively monitoring spore photoproduct repair mediated by spore photoproduct lyase during germination of uv-irradiated Bacillus subtilis spores.

The major DNA photoproduct formed upon uv irradiation of Bacillus subtilis spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, informally referred to as spore photoproduct (SP). A rapid separation technique for detecting and quantitating SP by HPLC was developed to replace traditional paper chromatography. Tritiated thymine and thymine-containing photoproducts from trifluoroacetic acid-hydrolyzed DNA purified from uv-irradiated cells or spores of B. subtilis were identified and isolated from paper chromatograms, subjected to HPLC on a Microsorb phenyl 5-microns column using 100% water as the mobile phase, and detected by scintillation counting of collected fractions. At a flow rate of 1.8 ml per minute, thymine-containing compounds eluted in the order thymine (T; 5.5 min), cis-syn cyclobutyl thymine-thymine dimers (TT; 7.5 min), cyclobutyl uracil-thymine dimers (UT, the acid breakdown product of cytosine-thymine (CT) dimers; 9.5 min), and SP (14.5 min). The method was used to quantitate the amount of SP produced upon irradiation of B. subtilis spores and to monitor repair of SP in vivo by the enzyme SP lyase during spore germination.