NMR Study of Repair Mechanism of DNA Photolyase by FAD-induced Paramagnetic Relaxation Enhancement

Abstract

Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 Å). Subsequently, a single-stranded DNA containing 13 C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 ؎ 3 Å. The FAD-induced PRE was also observed in the CPD photolyase-doublestranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.UV irradiation induces various lesions in DNA (1). The cyclobutane pyrimidine dimer (CPD) 1 is a major DNA lesion induced by UV light (2). CPD formation is closely linked to deleterious effects, including mutation, cell death, and skin cancer. Organisms have a variety of enzymes that recognize and repair DNA lesions. CPD photolyases repair CPDs using visible or near-UV light (3, 4). CPD photolyases induce in vivo photoreactivation, which prevents the deleterious effects of far-UV light (200 -300 nm) by the concurrent or subsequent exposure to near-UV-visible (300 -500 nm) light.CPD photolyases contain FAD as a catalytic cofactor. CPD photolyases repair the CPD by an electron transfer from photoactivated FAD to the CPD. The high quantum yield of the reaction ( ϭ 0.5-1.0) shows that CPD photolyases utilize light energy quite efficiently (5, 6). According to the theory proposed by Marcus (7), the distance and the relative orientation between donors and acceptors determines the efficiency of electron transfer reactions. Therefore, a structural analysis of the catalytic site of the enzyme is important for understanding the mechanism of the high efficiency of the reaction.The crystal structures of the CPD photolyases from Escherichia coli, Anacystis nidulans, and Thermus thermophilus HB8 were recently solved (8 -10). These structures show a similar global fold, and they have an FAD-containing cavity in the C-terminal helical domain. The crystal structure of the T. thermophilus CPD photolyase-thymine complex has also been solved (10). In this structure, the thymine molecule resided in the cavity, suggesting that this cavity forms the CPD-binding site. However, the structure of a CPD photolyase in complex with CPD-containing DNA has not yet been determined.Unpaired electrons induce paramagnetic relaxation enhancement (PRE) in NMR resonances from neighboring atoms in a distance-dependent manner, and the relaxation data are calculated with the Solomon-Bloembergen equation (11). PRE has been used for analyses of denatured proteins (12-14), protein-ligand comp...

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

“…PRE analysis for such systems is simple, since there are no pseudo-contact shifts, and Curie-spin relaxation that could potentially exhibit significant cross-correlation with other relaxation mechanisms (2,3), is negligible. Using this type of PRE data, macromolecular structures have been characterized for soluble proteins (4-10), protein-protein complexes (11)(12)(13)(14), protein-oligosaccharide complexes (15,16), protein-nucleic acid complexes (17)(18)(19)(20)(21), and membrane proteins (22,23). The PRE can also provide information relating to large-scale dynamics that accompany changes of paramagnetic center -1 H distances, for example in non-specific protein-DNA interactions (24,25) and interdomain motions (26).…”

Algal photoreceptors: in vivo functions and potential applications

“…They are activated by blue light and contain FAD as the catalytic chromophore and either methenyltetrahydrofolate (MTHF) or 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) as the second chromophore (Sancar 1994(Sancar , 2003. During last 1 3 few years, additional antenna cofactors such as FMN, FAD and 6,7-dimethyl-8-ribityl-lumazine have been identified in photolayses (Geisselbrecht et al 2012;Klar et al 2006;Ueda et al 2004). …”

Identification and Characterization of a Second Chromophore of DNA Photolyase from Thermus thermophilus HB27

“…2, trace 4), suggesting that the enzyme was in the FADH ⅐ form. NMR spectra of the enzyme with FADH ⅐ , in which the resonances from the atoms nearby FADH ⅐ had disappeared because of the FAD-induced paramagnetic relaxation enhancement (17), also suggested that Ͼ90% of the enzyme was in the FADH ⅐ form. However, the absorbance around 450 nm was also partially regenerated.…”

“…These structures share a similar overall fold consisting of an ␣/␤ domain and a helical domain. Our previous NMR analysis revealed that the CPD is bound to a FAD-containing cavity in the helical domain (17,18). The crystal structures of the CPD photolyases from E. coli and A. nidulans contain MTHF and 8-HDF, respectively, in the ␣/␤ domain (12,13,15,16), and the second chromophores are ϳ15 Å away from the FAD.…”