The native cyclobutane pyrimidine dimer photolyase of rice is phosphorylated.
The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containing DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54- and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia coli was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54- and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chromophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. coli-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. coli-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice.
Teranishi
Mika
M
Department of Environmental Life Sciences , Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. tera@ige.tohoku.ac.jp
Nakamura
Kentaro
K
Morioka
Hiroshi
H
Yamamoto
Kazuo
K
Hidema
Jun
J
D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D004255:Deoxyribodipyrimidine Photo-Lyase