An assay for DNA photolyases using non-radioactive DNA and restriction enzymes.
Restriction enzymes, such as Eco RI, Hind III, etc., which have a potential pyrimidine dimer site in their recognition sequence, fail to cleave DNA if their recognition site is modified by the formation of pyrimidine dimers as a result of UV irradiation of DNA (J. E. Cleaver, J. Mol. Biol., 170 (1983) 305-317). We have made use of this functional property of restriction enzymes to develop a rapid and sensitive assay for DNA photolyases. UV-irradiated plasmid pBR322 DNA is only partially digested when incubated with a single site enzyme Hind III even at high concentration (20 units (micrograms DNA)-1). The amount of DNA not cleaved by Hind III is determined by agarose gel electrophoresis. The effect of UV irradiation is reversed by the photoreactivation of DNA. The decrease in the amount of Hind III-resistant DNA on treatment with photolyase gives a measure of the enzymatic activity of the photolyase preparation. The advantage of using non-radioactive DNA and the high speed and simplicity of this assay make it especially suitable for use in the purification of photolyases.
Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Bombay, India.
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Dutta
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Hejmadi
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Verma
D001665:Binding Sites; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000528:radiation effects*; D004278:DNA, Superhelical / Q000378:metabolism*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000032:analysis*; D004307:Dose-Response Relationship, Radiation; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006193:Haemophilus influenzae / Q000235:genetics; D007700:Kinetics; D010957:Plasmids; D011740:Pyrimidine Dimers / Q000378:metabolism; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D011740:Pyrimidine Dimers; D004262:DNA Restriction Enzymes; D004255:Deoxyribodipyrimidine Photo-Lyase