Abstract
The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm(-1) bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm(-1) band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N5-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N3-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase.
Highlights
Irradiation of organisms with ultraviolet light causes damage to cellular DNA by inducing dimer formation between adjacent pyrimidine bases
Enzyme Activity—The time course curves for dimer repair by 2 M (6-4) and cyclobutane pyrimidine dimer (CPD) photolyases in the presence of 50 M UVdamaged oligo(dT)8 are shown in Fig. 4 (A and B, respectively)
Absorption Spectra of Photolyase Complexed with DNA Lesions—Immediately after purification, most of the (6-4) and CPD photolyases were in the neutral semiquinoid form with FADH° (Fig. 5, A and C, respectively)
Summary
Irradiation of organisms with ultraviolet light causes damage to cellular DNA by inducing dimer formation between adjacent pyrimidine bases. The resonance Raman spectra of the oxidized (6-4) and CPD The substrate-induced frequency shifts of FADox in (6-4) and photolyases are shown in Fig. 7 (A and B, respectively). The signal-to-noise ratio of the Interactions between UV-damaged DNA and the Isoallox-Raman spectra for the reconstituted oxidized CPD photolyase azine Ring of FAD—Fig. 6 and Table 1 show that the marker was not as high as that for the oxidized (6-4) photolyase, the band of the neutral semiquinoid flavin at 1607 cmϪ1 in CPD
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