Identification of oligothymidylates as new simple substrates for E. coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay

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Abstract
Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min) for the repair of dimers in oligothymidylates [oligo(dT)n] containing 4-18 thymine residues. This rate is identical with that observed with polythymidylate and with native DNA. The enzyme exhibits a similar high affinity with oligomers containing 7 or more thymine residues. A decrease in affinity is detectable with oligo(dT)n when n = 4-6. The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 .mu.M where the observed repair rate is 43% of the turnover number observed with the higher homolog. Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n .gtoreq. 9. Photolyase can repair internal dimers and dimers at a 5'' end where the terminal ribose is phosphorylated but not at unphosphorylated 5'' or 3'' ends. The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers. The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 [p(dT)2] since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3'' end. That the enzyme can repair dimers in short-chain, single-stranded analogs for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer. Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as a basis for a rapid spectrophotometric assay with a lower detection limit .apprx. 150 pmol of dimer repaired.