Temperature-sensitive photoreactivation of cyclobutane thymine dimer in soybean.
UV radiation induces the formation of two classes of photoproducts in DNA, the cyclobutane pyrimidine dimer (CPD) and the pyrimidine 6-4 pyrimidone photoproduct. CPDs in plants are repaired by class II CPD photolyase via a UV-A/blue light-dependent mechanism. The genes for the class II CPD photolyase have been cloned from higher plants such as Arabidopsis, Cucumis sativus (cucumber), Oryza sativa (rice) and Spinacia oleracea (spinach). Flavin adenine dinucleotide (FAD) has been identified as a cofactor. Here we report the isolation and characterization of the CPD photolyase cDNA from soybean (Glycin max). The sequence of amino acids predicted from the cDNA sequence was highly homologous to sequences of higher plant class II CPD photolyases. When the cDNA was expressed in a photolyase-deficient Escherichia coli, photoreactivation activity was partially restored by illumination with a fluorescent light. The purified enzyme showed CPD binding and light-dependent photoreactivation activities in vitro. When soybean CPD photolyase was heat-treated in vitro from 25 degrees C to 45 degrees C for 3 min, thymine dimer-binding activity and photoreactivation activity were decreased, and FAD was released from the enzyme. On the other hand, when the enzyme-CPD complex was heat-treated, photoreactivation activity was stable. We argue that FAD in the soybean CPD photolyase is labile for temperature, but once the enzyme-CPD complex has formed, FAD becomes tightly bound to the enzyme or complex.
Yamamoto
Ayumi
A
Graduate School of Life Sciences, Tohoku University, Japan.
Tanbir
Najrana
N
Hirouchi
Tokuhisa
T
Teranishi
Mika
M
Hidema
Jun
J
Morioka
Hiroshi
H
Yamamoto
Kazuo
K
D018076:DNA, Complementary / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008027:Light; D011740:Pyrimidine Dimers / Q000528:radiation effects*; D013025:Glycine max / Q000378:metabolism*; D013696:Temperature
D018076:DNA, Complementary; D011740:Pyrimidine Dimers; D005182:Flavin-Adenine Dinucleotide