| Autor: |
Yamada D; Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.; Graduate School of Science, University of Hyogo, Hyogo 678-1297, Japan., Yamamoto J; Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan., Getzoff ED; Department of Integrative Structural and Computational Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, United States.; Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States., Iwata T; Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.; Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan., Kandori H; Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan. |
| Abstrakt: |
Photolyases (PHRs) repair ultraviolet (UV)-induced DNA photoproducts into normal bases. In this study, we measured the conformational changes upon photoactivation and photorepair processes of a PHR and its specific substrates, (6-4)PHR and a pyrimidine(6-4)pyrimidone photoproduct ((6-4)PP), by light-induced difference Fourier transform infrared (FT-IR) spectroscopy. The single-stranded DNA with (6-4)PP (ss(6-4)PP) was used as a substrate and the resultant FT-IR spectra were compared with the previous results on double-stranded DNA with (6-4)PP (ds(6-4)PP). In the excess amount of substrate to the enzyme, different ss(6-4)PP photorepair FT-IR signals were obtained in an illumination time-dependent manner. As reported for ds(6-4)PP, the early stages of the photoreaction involve the changes in the ss(6-4)PP only, while the late stages of the reaction involve the ss(6-4)PP repair-associated changes and dissociation from (6-4)PHR. From these spectra, difference spectra originating from the binding/dissociation spectrum were extracted. The signals of the C═O stretches of (6-4)PP and repaired thymines in the single- and double-stranded DNA were tentatively assigned. The C═O stretches of (6-4)PP were observed at frequencies that reflect single- and double-stranded DNA environments in aqueous solution, reflecting the different hydrogen-bonding environments. The conformational changes of PHR upon binding of ss(6-4)PP and ds(6-4)PP were similar, suggesting that the conformational change is limited to the (6-4)PP binding pocket region. We interpreted that ds(6-4)PP may be bound together without any special mechanism for flipping out. |
