Biochemical and biological properties of DNA photolyases derived from utraviolet-sensitive rice cultivars.
Class I and class II CPD photolyases are enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We identified three amino acid changes, Gln126Arg, Gly255Ser, and Gln296His, among which Gln but not His at 296 is important for complementing phr-defective E. coli, binding UV-damage in E. coli, and binding thymine dimers in vitro. The photolyase with Gln at 296 has an apoenzyme:FAD ratio of 1 : 0.5 and that with His at 296 has an apoenzyme:FAD ratio of 1 : 0.12-0.25, showing a role for Gln at 296 in the binding of FAD not in the binding of thymine dimer. Concerning Gln or Arg at 126, the biochemical activity of the photolyases purified from E. coli and complementing activity for phr-defective E. coli are similarly proficient. However, the sensitivity to UV of cultivars differs depending on whether Gln or Arg is at 126. The role of Gln and Arg at 126 for photoreactivation in rice is discussed.
Yamamoto
Ayumi
A
Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
Hirouchi
Tokuhisa
T
Mori
Tamiki
T
Teranishi
Mika
M
Hidema
Jun
J
Morioka
Hiroshi
H
Kumagai
Tadashi
T
Yamamoto
Kazuo
K
D019943:Amino Acid Substitution / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D009154:Mutation / Q000235:genetics; D012275:Oryza / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects*; D011740:Pyrimidine Dimers / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014466:Ultraviolet Rays
D018744:DNA, Plant; D011740:Pyrimidine Dimers; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D004255:Deoxyribodipyrimidine Photo-Lyase