Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA.
The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (lambda greater than 300 nm). However, the purified PRE from E. coli has no well-defined absorption band for lambda greater than 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 wavelength less than 300 nm. The hyperchromicity for lambda greater than 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (lambda greater than 300 nm) and hypochromicity (lambda less than 300 nm) can be reversed by irradiation of 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.
D004269:DNA, Bacterial / Q000378:metabolism / Q000528:radiation effects*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008190:Lyases / Q000378:metabolism*; D046911:Macromolecular Substances; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase