title: Binding of Escherichia coli DNA photolyase to UV-irradiated DNA.
abstract: Escherichia coli DNA photolyase is a flavoprotein which catalyzes the photomonomerization of pyrimidine dimers produced in DNA by UV irradiation. In vivo, the enzyme acts by a two-step mechanism: it binds to dimer-containing DNA in a light-independent reaction and upon exposure to 300-500-nm light breaks the cyclobutane ring and dissociates from the substrate. Using photolyase purified to homogeneity, we have investigated in vitro the first step of the reaction, DNA binding; enzyme-DNA complex formation was quantitated by the nitrocellulose filter binding assay. We find that the enzyme binds specifically to UV-irradiated DNA regardless of whether the DNA is in the superhelical, open circular, or linear form or whether the DNA is single or double stranded. The binding reaction is optimum at a NaCl concentration of 125 mM and at pH 7.5. Although photolyase is retained by the nitrocellulose filters with near 100% efficiency, the binding efficiency of a single enzyme-substrate complex is about 0.34. The complexes can be dissociated by exposing them to photoreactivating light either in solution or on the filter.
mesh_terms: D004269:DNA, Bacterial / Q000378:metabolism / Q000528:radiation effects*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D010957:Plasmids / Q000528:radiation effects*; D011485:Protein Binding; D011740:Pyrimidine Dimers / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014316:Tritium; D014466:Ultraviolet Rays
chemical_list: D004269:DNA, Bacterial; D011740:Pyrimidine Dimers; D014316:Tritium; D012965:Sodium Chloride; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase