title: FTIR study of light-dependent activation and DNA repair processes of (6-4) photolyase.
abstract: The UV component of sunlight threatens all life on the earth by damaging DNA. The photolyase (PHR) DNA repair proteins maintain genetic integrity by harnessing blue light to restore intact bases from the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPD), and (6-4) photoproducts ((6-4) PPs). The (6-4) PHR must catalyze not only covalent bond cleavage between two pyrmidine bases but also hydroxyl or amino group transfer from the 5'- to 3'-pyrimidine base, requiring a more complex mechanism than that postulated for CPD PHR. In this paper, we apply Fourier transform infrared (FTIR) spectroscopy to (6-4) PHR and report difference FTIR spectra that correspond to its photoactivation, substrate binding, and light-dependent DNA repair processes. The presence of DNA carrying a single (6-4) PP uniquely influences vibrations of the protein backbone and a protonated carboxylic acid, whereas photoactivation produces IR spectral changes for the FAD cofactor and the surrounding protein. Difference FTIR spectra for the light-dependent DNA damage repair reaction directly show significant DNA structural changes in the (6-4) lesion and the neighboring phosphate group. Time-dependent illumination of samples with different enzyme:substrate stoichiometries successfully distinguished signals characteristic of structural changes in the protein and the DNA resulting from binding and catalysis.
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Iwata
authors.forename: Junpei
authors.initials: J
authors.lastname: Yamamoto
authors.forename: Kenichi
authors.initials: K
authors.lastname: Hitomi
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Todo
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Getzoff
authors.forename: Hideki
authors.initials: H
authors.lastname: Kandori
mesh_terms: D000818:Animals; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000378:metabolism; D008027:Light; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011740:Pyrimidine Dimers / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013472:Sunlight; D014466:Ultraviolet Rays; D014981:Xenopus
chemical_list: D011740:Pyrimidine Dimers; D011743:Pyrimidines; C030986:pyrimidine; D010100:Oxygen