title: Detection of distinct α-helical rearrangements of cyclobutane pyrimidine dimer photolyase upon substrate binding by Fourier transform infrared spectroscopy.
abstract: Photolyases (PHRs) utilize near-ultraviolet (UV)-blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer PHR (CPD-PHR) from Escherichia coli binds flavin adenine dinucleotide (FAD) as a cofactor and 5,10-methenyltetrahydrofolate as a light-harvesting pigment and specifically repairs CPD lesions. By comparison, a second photolyase known as (6-4) PHR, present in a range of higher organisms, uniquely repairs (6-4) PPs. To understand the repair mechanism and the substrate specificity that distinguish CPD-PHR from (6-4) PHR, we applied Fourier transform infrared (FTIR) spectroscopy to bacterial CPD-PHR in the presence or absence of a well-defined DNA substrate, as we have studied previously for vertebrate (6-4) PHR. PHRs show light-induced reduction of FAD, and photorepair by CPD-PHR involves the transfer of an electron from the photoexcited reduced FAD to the damaged DNA for cleaving the dimers to maintain the DNA's integrity. Here, we measured and analyzed difference FTIR spectra for the photoactivation and DNA photorepair processes of CPD-PHR. We identified light-dependent signals only in the presence of substrate. The signals, presumably arising from a protonated carboxylic acid or the DNA substrate, implicate conformational rearrangements of the protein and substrate during the repair process. Deuterium exchange FTIR measurements of CPD-PHR highlight potential differences in the photoactivation and photorepair mechanisms in comparison to those of (6-4) PHR. Although CPD-PHR and (6-4) PHR appear to exhibit similar overall structures, our studies indicate that distinct conformational rearrangements, especially in the α-helices, are initiated within these enzymes upon binding of their respective DNA substrates.
authors: Department of Frontier Materials, Nagoya Institute of Technology, Nagoya 466-8555, Japan.
authors: I M Mahaputra
authors: IM
authors: Wijaya
authors: Yu
authors: Y
authors: Zhang
authors: Tatsuya
authors: T
authors: Iwata
authors: Junpei
authors: J
authors: Yamamoto
authors: Kenichi
authors: K
authors: Hitomi
authors: Shigenori
authors: S
authors: Iwai
authors: Elizabeth D
authors: ED
authors: Getzoff
authors: Hideki
authors: H
authors: Kandori
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011740:Pyrimidine Dimers / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D011740:Pyrimidine Dimers; D005182:Flavin-Adenine Dinucleotide; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase