Abstract
Cyclobutane pyrimidine dimer (CPD) is a major type of UV-induced DNA damage. CPD photolyase is a crucial factor for determining the sensitivity of plant to UVB. CPD photolyases have been reported in many organisms and classified into two classes, class I and class II, based on their similarity of amino acid sequence. Class I CPD photolyases have been studied well. However, little is known about class II CPD photolyase. To characterize rice class II CPD photolyase, the enzyme from rice leaves was partially purified by 4 purification steps. At the final purification step, the enzyme bound to CPD-containing DNA-conjugated magnetic beads were released by blue-irradiation. Specific activity of the purified enzyme was 8,100-fold higher than that of crude extract. SDS-PAGE of the purified enzyme showed existence of two proteins of about 54- and 56-kDa. Western blot analysis using anti-rice CPD photolyase antibody and TOF-MS analysis showed that both proteins were CPD photolyase. In the transgenic plant bearing the cDNA of rice CPD photolyase in the sense orientation, the revel of both 54- and 56-kDa proteins were significantly higher than those in wild-type plant. It is thus speculated that the difference between 54- and 56-kDa protein is due to post-translational modification. Furthermore, the CPD photorepair activity by the native rice CPD photolyase was significant higher than that of E. coli-expressed rice CPD photolyase, which showed one band about 55-kDa on SDS-PAGE. These results suggested that native CPD photolyase is different from E. coli-expressed protein.
