Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA

Abstract

The photoreactivation enzyme PRE monomerizes pyrimidine dimers in DNA in a light requiring reaction (.lambda. [wavelength] > 300 nm). The purified PRE from E. coli has no well-defined absorption band for .lambda. > 300 nm. Absorption difference spectroscopy showed that when PRE is mixed with UV-irradiated DNA, new absorption appears in the spectral region required for catalysis. There is a concomitant decrease in the absorption of the mixture for .lambda. < 300 nm. The hyperchromicity for .lambda. > 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (.lambda. > 300 nm) and hypochromicity (.lambda. < 300 nm) can be reversed by irradiation at 365 nm with identical 1st-order kinetics. The molar extinction coefficient of the new absorption was 6900 .+-. 1400 at 350 nm. The PRE from E. coli apparently does not possess a distinct chromophore which by itself is entirely responsible for the absorption of photoreactivating light. Instead, new absorption results when PRE binds its substrate, dimer-containing DNA.