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 rice to UVB. We previously reported that the native rice CPD photolyase was phosphorylated. To investigate the effect of phosphorylation on CPD photolyase function, we tried to determine the phosphorylation site of rice CPD photolyase using site-directed mutagenesis method. We previously reported that the E. coli-expressed rice CPD photolyase was not phosphorylated. Thus, we used an insect cell-free translation system, which has eukaryotic posttranslational modification machinery. We found that rice CPD photolyase, which was synthesized using insect cell-free translation system, was phosphorylated. Then, we constructed plasmids carrying the genes encoding rice CPD photolyase which replaced the putative phosphorylation site with alanine residue by site-directed mutagenesis method. The point-mutated proteins were synthesized using the plasmids by an insect cell-free translation system. In this study, we discuss about the effects of phosphorylation of CPD photolyase on its function.
