title: Role of the carbonyl group of the (6-4) photoproduct in the (6-4) photolyase reaction.
abstract: The (6-4) photoproduct, which is one of the major UV-induced DNA lesions, causes carcinogenesis with high frequency. The (6-4) photolyase is a flavoprotein that can restore this lesion to the original bases, but its repair mechanism has not been elucidated. In this study, we focused on the interaction between the enzyme and the 3' pyrimidone component of the (6-4) photoproduct and prepared a substrate analogue in which the carbonyl group, a hydrogen-bond acceptor, was replaced with an imine, a hydrogen-bond donor, to investigate the involvement of this carbonyl group in the (6-4) photolyase reaction. UV irradiation of oligodeoxyribonucleotides containing a single thymine-5-methylisocytosine site yielded products with absorption bands at wavelengths longer than 300 nm, similar to those obtained from the conversion of the TT site to the (6-4) photoproduct. Nuclease digestion, MALDI-TOF mass spectrometry, and the instability of the products indicated the formation of the 2-iminopyrimidine-type photoproduct. Analyses of the reaction and the binding of the (6-4) photolyase using these oligonucleotides revealed that this imine analogue of the (6-4) photoproduct was not repaired by the (6-4) photolyase, although the enzyme bound to the oligonucleotide with considerable affinity. These results indicate that the carbonyl group of the 3' pyrimidone ring plays an important role in the (6-4) photolyase reaction. On the basis of these results, we discuss the repair mechanism.
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Junpei
authors.initials: J
authors.lastname: Yamamoto
authors.forename: Kenichi
authors.initials: K
authors.lastname: Hitomi
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Hayashi
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Getzoff
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000528:radiation effects; D024202:Electrophoretic Mobility Shift Assay; D006860:Hydrogen Bonding / Q000528:radiation effects; D006868:Hydrolysis / Q000528:radiation effects; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D055668:Photochemical Processes / Q000528:radiation effects; D011744:Pyrimidinones / Q000737:chemistry / Q000528:radiation effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays; D014982:Xenopus laevis
chemical_list: D009838:Oligodeoxyribonucleotides; D011744:Pyrimidinones; D004255:Deoxyribodipyrimidine Photo-Lyase