Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VII. Photolysis of enzyme-substrate complexes in vitro

Abstract

The extent of complexing of the repairable ultraviolet lesions in Haemophilus influenzae transforming DNA with yeast photoreactivating enzyme in the dark can be determined by applying intense light flashes, which cause repair of essentially all lesions complexed. With full complexing of repairable lesions the kinetics of photoreactivation by steady illumination reflect only the photochemical reaction occurring in the enzyme-substrate complex. Complexes are heterogeneous with respect to their rates of photolysis, described by the first-order rate constant k 3. At early stages of repair k 3 = k p I, where I is the light intensity and k p is the photolysis constant. The measured value of k p permits calculation of the product ϵΦ, where ϵ is the molar extinction coefficient for the complex, and Φ is the quantum yield for photolysis, giving an absolute action spectrum. In the region of most effective wavelengths (355–385 nm), ϵ > 10 4 liter·mole −1·cm −1, and Φ > 0.1, with the possibility that Φ ∼ 1. k p is independent of temperature and ionic strength over a range producing large effects on the rate of complex formation, but does increase with pH over the range 6.7–7.4. For the slower repair of a minority of lesions the measured value of k p depends somewhat on intensity and continuity of illumination, suggesting some differences not yet understood. At the usual illumination intensities used for photoreactivation k 3 is greater than k 2 the rate constant for dark dissociation of complexes.