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内容記述 |
Ultraviolet light radiation in the wavelength range between 260 and 320 nm causes<br />damage to DNA by forming the dimerization of adjacent pyrimidines in the same DNA<br />strand. This covalentry linked dimer influences the replication and transcription, and leads<br />to cell death or skin cancer. Most (70-80%) of UV-induced DNA lesions is cyclobutane<br />pyrimidine dimer (CPD) and, a lesser extent (20-30%) is (6-4) photoproduct. These two<br />major types of UV-damaged DNA are repaired under illumination with near-UV/visible<br />light by CPD photolyase and (6-4) photolyase, respectively. The both photolyases contain<br />flavin adenine dinucleotide (FAD) as an essential cofactor for DNA repairing. The structure<br />and catalytic mechanism of CPD photolyase have been extensively studied. However, its<br />detailed structure of the active site containing CPD is uncertain until now. The structure of <br />(6-4) photolyase is less well studied. The genes for this enzyme exhibit a sequence<br />similarity to CPD photolyase, especially in the FAD binding sites. Such high similarity<br />indicates a similar structure and reaction mechanism in two photolyases. Unexpectedly,<br />(6-4) photolyase presents a much lower quantum yield compared to that of CPD photolyase,<br />indicating the differences in structure and reaction mechanism between (6-4) and CPD<br />photolyase. Such precise differences between CPD photolyase and (6-4) photolyase<br />regarding substrate binding and DNA repair needs to be clarified.<br /> To investigate the unclear structure and environment of the active site in (6-4)<br />photolyase, we measured resonance Raman spectra of (6-4) photolyase having neutral<br />semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by<br />excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the<br />first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606<br />cm<sup>-1</sup> in H<small>2</small>O. whose frequency is the lowest among various flavoenzymes, apparently splits<br />into two comparable bands at 1594 and 1608 cm<sup>-1</sup> in D<small>2</small>O, and similarly that at 1522 cm<sup>-1</sup> in <br />H<small>2</small>O does into three bands at 1458, 1508, and 1536 cm<sup>-1</sup> in D<small>2</small>O. This D<small>2</small>O effect was <br />recognized only after being oxidized once and photoreduced to form a semiquinone again,<br />but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were<br />observed at significantly low frequencies (1621, 1576 cm<sup>-1</sup>)and with band splittings<br />(1508/1493, 1346/1320 cm<sup>-1</sup>). These Raman spectral characteristics indicate strong H-bonding<br />interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in<br />benezene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.<br /> To clarify the structure of active site upon substrate binding and the mechanism of DNA repair, we<br />examined the resonance Raman spectra of complexes between damaged DNA and the neutral<br />semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral<br />semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the<br />benzene ring of flavin adenine dinucleotide (FAD), were shifted to lower freauencies upon binding of <br />damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the<br />benzene ring of FAD directly but that (6-4) photoproduct does not. Bands II and VII of the oxidized<br />flavin and the 1398/1391 cm<sup>-1</sup> bands of the neutral semiquinoid flavin, which may reflect the bending of<br />the U-shaped FAD, were altered upon substrate binding suggesting that CPD and (6-4) photoproduct<br />interact with the adenine ring of FAD, When substrate is bound, there is an upshiftesd 1528 cm<sup>-1</sup> band of<br />the neutral semiquinoid flavin in CPD photolyase, indicating a weekend hydrogen bond at N5-H of<br />FAD, and in (6-4) photolyase, band X is downshifted, indicating a strengthened hydrogen bond at N3-H<br />of FAD. These Raman spectra led us to conclude that the two photolyases have different electron <br />transfer mechanisms as well as different hydrogen bonding environments, which account for the higher<br />redox potential of CPD photolyase.<br /> This work revealed that the FAD in (6-4) photolyase is characterized by an electron<br />localized structure, and binds to the protein in a fairly hydrophobic and strong hydrogen <br />bonding environment, Specially, a stronger H-bonding at N5-H of FAD was identified for <br />(6-4) photolyase, which may result in the low quantum yield for DNA-repair of this<br />enzyme. Besides, UV-damaged DNA contacts the benzene ring of FAD only in CPD<br />photolyase and the adenine ring of FAD in both photolyases. These structures indicate that<br /> the election transfer during DNA -repair between isoalloxazine and UV-damaged DNA in <br />CPD photolyase is direct, whereas that in (6-4) photolyase is not direct and bridged by<br />adenine. |
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