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Figure 1. UV-damaged DNA and its photorepair by photolyases. (A) Photorepair reaction scheme of CPDs and (6–4)PPs by CPD-PL and (6–4)PL, respectively. (B and C) Close-up views of the complex structure of Methanosarcina mazei class II CPD-PL (B) and Drosophila melanogaster (6–4)PL (C). The coordinates were taken from Protein Data Bank with the accession numbers of 2XRZ (17) and 3CVU (18). (D) Sequence alignment of a selected region among CPD-PL and (6–4)PL. Abbreviations: Ec, Escherichia coli; An, Anacystis nidulans; At, Arabidopsis thaliana; Os, Oryza sativa; Mm, Methanosarcina mazei; Dm, Drosophila melanogaster; Xl, Xenopus laevis.
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Figure 2. Binding of the WT and mutants of Xl64 to the (6–4)PP containing substrate. (A) Selected view of the structure of Dm64 after the QM/MM optimization (see Materials and Methods). (B) UV survival assays of bacteria producing the WT or mutants of Xl64. Null means no production of Xl64 from the empty vector. The experiments were independently repeated three times (n = 3), and statistical analysis was carried out by the Student's t-test, where the significance was set to P < 0.01. Asterisks indicated the P-values of 0.0048 (*) and 0.0033 (**). (C) Electromobility shift assays of WT-Xl64. The upper bands represent the complex between 32P-DNA and the enzyme. Aliquots (0.5 nmol) of the 32P-labeled double-stranded 49-mer oligonucleotides were incubated with WT-Xl64 for 30 min at increasing concentrations of 0, 0.5, 1, 2, 5, 10, 20, 50, 100, 150 and 200 nM (lanes 1–11), and free- and bound-DNA were separated on a 5% polyacrylamide gel. The experiments were performed in triplicate, and the band intensities were quantified. (D) Binding of the WT (black square), Q407A (blue circle), R410A (red triangle), and R410K (open red triangle) of Xl64 to the (6–4)PP-containing double-stranded DNA. The complex concentrations were calculated from the quantified band intensities, and the mean values were plotted against enzyme concentrations. Their fitting curves are also shown.
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Figure 3. Photorepair of (6–4)PP by the WT and mutants of Xl64. (A) Spectral bleaching of the (6–4)PP band upon photorepair of the single-stranded 8-mer substrate by WT-Xl64. The inset represents the time course of the absorption change at 325 nm upon 384-nm illumination. (B) Photorepair of the 8-mer substrate by the WT (black square) and the Xl64 mutants (Q407A: blue circle, R410A: red triangle, and R410K: red open triangle). The numbers of the repaired substrate per enzyme were plotted against the numbers of the incident photons absorbed by FADH–.
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Figure 4. Pair interaction energy decomposition analysis (PIEDA) in the FMO calculation. (A) Chemical structures of the fragments in the FMO calculation for PLWT. In the calculation, the molecules were divided into eight fragments; the Gln residue (fragment 1), the Arg residue (fragment 2), dGMP 5′ next to the (6–4)PP (fragment 3), the 5′ side deoxyribose moiety of the (6–4)PP with the internucleoside phosphate within the (6–4)PP (fragment 4), the 3′ side deoxyribose moiety of the (6–4)PP with the 3′ flanking phosphate (fragment 5), the base moiety of the (6–4)PP (fragment 6), the deoxyribose moiety of the dG 3′ flanking the (6–4)PP (fragment 7), and the base moiety of the 3′ dG. In the case of the mutants (PLR421A and PLR421K), the fragment 2 was replaced with the corresponding amino acid residue. The IFIEs for these fragments were calculated at the FMO2-MP2/6-31G(d,p) level with the PIEDA option. Red and blue dotted lines represent remarkable electrostatic and dispersive interactions, respectively. Note that we simulated the ideal contribution of the electrostatic and dispersive interaction energies without taking into account the solvation, in order to exclude the solvation-induced stabilizing effect. (B) Heat maps of the electrostatic and dispersion energies (Ees and Edisp, shown in red and blue, respectively) between the two individual fragments in PLWT, PLR421A, or PLR421K. The detailed values of the simulated interaction energies are summarized in Supplementary Tables S4–S6. In PIEDA, Ees and Edisp for the fragment pair originally linked through a covalent bond (e.g. fragments 4 and 5) were found to be largely negative (see Supplementary Tables S4–S6). These interactions are shown in gray, not to mix them up with the spatial interactions in the fragments of interest. (C) Selected interaction energies. Full lists of the interaction energies are summarized in Supplementary Tables S4–S6.
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Figure 5. Binding and photorepair properties of WT-Xl64 using chemically modified substrates. (A) Structures of the DHT and AP substrates. The 5′ terminus of each oligonucleotide for the anisotropy measurement was modified with the C3 amino linker on a DNA synthesizer, followed by labeling with TAMRA, while the DHT substrates used for the DNA repair assays were unlabeled, and had the intact 5′-hydroxy group. (B) Fluorescence anisotropy measurements of the fluorescently labeled DHT-1 (black), DHT-2 (blue), DHT-3 (red), and DHT-4 (green) upon titration with increasing amounts of WT-Xl64. The measurements were performed independently in triplicate, and the mean ± SD values are shown. (C) Fluorescence anisotropy measurements of fluorescently labeled (6–4)T8 (black square), 3AP (red triangle), and 5AP (blue triangle) upon titration with increasing amounts of WT-Xl64. (D) Photorepair of the DHT substrates by WT-Xl64.
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Figure 6. Important interactions for and their roles in the stabilization of the repair-active DNA-(6–4)PL complex.
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Figure 7. Binding of the animal CRY-type mutant of Xl64 to the (6–4)PP-containing substrates. (A) Sequence alignment of the (6–4)PLs and animal CRYs. The arginine and histidine residues in question are highlighted in red and blue, respectively. Abbreviations: Dr, Danio rerio; Er, Erithacus rubecula; Mm, Mus musculus; Hs, Homo sapiens. (B) Fluorescence anisotropy measurements of the fluorescently labeled (6–4)T8 (black square), DHT-1 (black circle), and DHT-3 (red circle) upon titration with increasing amounts of R410H-Xl64. The measurements were performed independently in triplicate, and the mean ± SD values are shown. (C) Dissociation constants (Kd) for the binding of R410H-Xl64 to the tested substrates.
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