Fourier-Transform Infrared Study of the Photoactivation Process of <i>Xenopus</i> (6–4) Photolyase

Photolyases (PHRs) are blue light-activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The flavin adenine dinucleotide (FAD) chromophore of PHRs has four different redox states: oxidized (FAD^ox), anion radical (FAD^•–), neutral radical (FADH^•), and fully reduced (FADH^–). We combined difference Fourier-transform infrared (FTIR) spectroscopy with UV–visible spectroscopy to study the detailed photoactivation process of <i>Xenopus</i> (6–4) PHR. Two photons produce the enzymatically active, fully reduced PHR from oxidized FAD: FAD^ox is converted to semiquinone via light-induced one-electron and one-proton transfers and then to FADH^– by light-induced one-electron transfer. We successfully trapped FAD^•– at 200 K, where electron transfer occurs but proton transfer does not. UV–visible spectroscopy following 450 nm illumination of FAD^ox at 277 K defined the FADH^•/FADH^– mixture and allowed calculation of difference FTIR spectra among the four redox states. The absence of a characteristic CO stretching vibration indicated that the proton donor is not a protonated carboxylic acid. Structural changes in Trp and Tyr are suggested by UV–visible and FTIR analysis of FAD^•– at 200 K. Spectral analysis of amide I vibrations revealed structural perturbation of the protein's β-sheet during initial electron transfer (FAD^•– formation), a transient increase in α-helicity during proton transfer (FADH^• formation), and reversion to the initial amide I signal following subsequent electron transfer (FADH^– formation). Consequently, in (6–4) PHR, unlike cryptochrome-DASH, formation of enzymatically active FADH^– did not perturb α-helicity. Protein structural changes in the photoactivation of (6–4) PHR are discussed on the basis of these FTIR observations

H-NMR spectra ( and ) and NOE difference spectra ( and ) of compounds and

<p><b>Copyright information:</b></p><p>Taken from "Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction"</p><p>Nucleic Acids Research 2006;34(5):1540-1551.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409675.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> ( and ) the major isomer; ( and ) the minor isomer. The H6 resonance was saturated in the NOE experiments

Competition of the 13 bp substrate duplexes and the Tg-containing duplexes in the Endo III reaction

<p><b>Copyright information:</b></p><p>Taken from "Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction"</p><p>Nucleic Acids Research 2006;34(5):1540-1551.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409675.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The P-labeled substrates without fluorine were incubated with Endo III in the presence of the competitors containing Tg. The amounts of the nicked products (standardized to those without the competitors) were plotted against the concentrations of the competitor. Open circles, P-5-Tg + 5-Tg; filled circles, P-5-Tg + 5-Tg; open triangles, P-5-Tg + 5-Tg; filled triangles, P-5-Tg 5-Tg

Analysis of the 5-Tg () and 5-Tg () 13mers by MALDI-TOF mass spectrometry

<p><b>Copyright information:</b></p><p>Taken from "Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction"</p><p>Nucleic Acids Research 2006;34(5):1540-1551.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409675.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The calculated molecular weight is 3977.69

Formation of the covalent complex between T4 Endo V and a CPD-containing oligonucleotide duplex fluorinated at the 2′ position

<p><b>Copyright information:</b></p><p>Taken from "Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction"</p><p>Nucleic Acids Research 2006;34(5):1540-1551.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409675.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The 5′ and 3′ ends of the CPD-containing strand were P-labeled. After hybridization with the complementary strand, the duplexes were incubated with T4 Endo V at 30°C for 30 min, and the mixtures were subjected to 15% SDS–PAGE

Structural Changes of the Active Center during the Photoactivation of <i>Xenopus</i> (6–4) Photolyase

Photolyases (PHRs) repair the UV-induced photoproducts, cyclobutane pyrimidine dimer (CPD) or pyrimidine-pyrimidone (6–4) photoproduct [(6–4) PP], restoring normal bases to maintain genetic integrity. CPD and (6–4) PP are repaired by substrate-specific PHRs, CPD PHR and (6–4) PHR, respectively. Flavin adenine dinucleotide (FAD) is the chromophore of both PHRs, and the resting oxidized form (FAD^ox), at least under <i>in vitro</i> purified conditions, is first photoconverted to the neutral semiquinoid radical (FADH^•) form, followed by photoconversion into the enzymatically active fully reduced (FADH^–) form. Previously, we reported light-induced difference Fourier transform infrared (FTIR) spectra corresponding to the photoactivation process of <i>Xenopus</i> (6–4) PHR. Spectral differences between the absence and presence of (6–4) PP were observed in the photoactivation process. To identify the FTIR signals where these differences appeared, we compared the FTIR spectra of photoactivation (i) in the presence and absence of (6–4) PP, (ii) of ¹³C labeling, ¹⁵N labeling, and [¹⁴N]­His/¹⁵N labeling, and (iii) of H354A and H358A mutants. We successfully assigned the vibrational bands for (6–4) PP, the α-helix and neutral His residue(s). In particular, we assigned three bands to the CO groups of (6–4) PP in the three different redox states of FAD. Furthermore, the changed hydrogen bonding environments of CO groups of (6–4) PP suggested restructuring of the binding pocket of the DNA lesion in the process of photoactivation

Detection of Distinct α‑Helical Rearrangements of Cyclobutane Pyrimidine Dimer Photolyase upon Substrate Binding by Fourier Transform Infrared Spectroscopy

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 <i>Escherichia coli</i> 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

Substrate Assignment of the (6-4) Photolyase Reaction by FTIR Spectroscopy

Photolyases (PHRs) are DNA repair proteins that revert UV-induced photoproducts, either cyclobutane pyrimidine dimers (CPD) or (6-4) photoproducts (PPs), into normal bases to maintain genetic integrity. The (6-4) PHR must catalyze not only covalent bond cleavage but also hydroxyl or amino group transfer, yielding a more complex mechanism than that postulated for CPD PHR. Building upon recently established light-induced difference FTIR spectroscopy of <i>Xenopus</i> (6-4) PHR, we now utilize ¹⁵N_3′-labeled (6-4) PP to identify vibrational modes of (6-4) PP upon repair. We successfully assign two and three vibrational bands for the (6-4) PP and the repaired thymine, respectively. Thus, the present FTIR spectroscopy is sensitive enough to distinguish a single nitrogen atom (¹⁵N versus ¹⁴N) among >6700 atoms in the enzyme–substrate complex

Flavin Adenine Dinucleotide Chromophore Charge Controls the Conformation of Cyclobutane Pyrimidine Dimer Photolyase α‑Helices

Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apoprotein, and substrate, so that only the initial, ultrafast, photon–chromophore reaction and the final, slow, protein conformational change provide separate, nonoverlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening time scales still cannot be fully deconvoluted. We overcome the problem by using a novel method to selectively isotope-label the apoprotein but not the flavin adenine dinucleotide (FAD) cofactor. This allowed the Fourier transform infrared (FTIR) signals to be separated from the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive structure–function study by FTIR spectroscopy of the <i>Escherichia coli</i> cyclobutane pyrimidine dimer photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair, which revealed protein dynamics for both processes beyond simple one-electron reduction and ejection, respectively. The FTIR data suggest that the synergistic cofactor–protein partnership in CPD-PHR linked to changes in the shape of FAD upon one-electron reduction may be coordinated with conformational changes in the apoprotein, allowing it to fit the DNA substrate. Activation of the CPD-PHR chromophore primes the apoprotein for subsequent DNA repair, suggesting that CPD-PHR is not simply an electron-ejecting structure. When FAD is activated, changes in its structure may trigger coordinated conformational changes in the apoprotein and thymine carbonyl of the substrate, highlighting the role of Glu275. In contrast, during DNA repair and release processes, primary conformational changes occur in the enzyme and DNA substrate, with little contribution from the FAD cofactor and surrounding amino acid residues