Binding ofE. coliDNA photolyase to a defined substrate containing a single T< >T dimer

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Abstract
The E. coli DNA photolyase is a flavoprotein that catalyzes the photo-reversal of pyrimidine dimers. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and repairs the dimer upon absorbing a photon in the 300–600 nm range. The rate and equilibrium constants for the light-independent reaction were determined before, using randomly modified substrates that contained T<>T, T<>C and C<>C dimers in random sequence surrounding. In this paper we have determined these constants for a defined substrate (a 43 bp oligomer containing a T<>T dimer) using the gel retardation assay. We find that: (i) the equilibrium constant and the off rate obtained with this substrate by this technique are similar to those obtained with randomly modified DNA using filter binding and flash photolysis techniques, (ii) the off rate with the defined substrate is heterogenous indicating heterogeneity in the enzyme population or in the enzyme-substrate complexes, and (iii) the enzyme has 7.5 × 104−fold higher affinity for pyrimidine dimer compared to non-dimer DNA nucleotides.