The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (λ > 300 nm). However, the purified PRE from E. coli has no well-defined absorption band for λ > 300 nm. Using absorption difference spectroscopy, we show that when PRE is mixed with ultraviolet-irradiated DNA, new absorption appears in the spectral region required for catalysis. There is a concomitant decrease in the absorption of the mixture for wavelengths less than 300 nm. The hyperchromicity for λ > 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (λ > 300 nm) and hypochromicity (λ < 300 nm) can be reversed by irradiation at 365 nm with identical first-order kinetics. We estimate the molar extinction coefficient of the new absorption to be 6900 ± 1400 at 350 nm. We conclude that the PRE from E. coli 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.
ASJC Scopus subject areas