Effect of base, pentose, and phosphodiester backbone structures on binding and repair of pyrimidine dimers by Escherichia coli DNA photolyase.
Photolyases reverse the effects of UV light on cells by converting cyclobutane dipyrimidine photoproducts (pyrimidine dimers, Pyr mean value of Pyr) into pyrimidine monomers in a light-dependent reaction. Previous work has suggested that, based on substrate preference, there are two classes of photolyase: DNA photolyase as exemplified by the Escherichia coli enzyme, and RNA photolyases found in plants such as Nicotiana tabacum and Phaseolus vulgaris. In experiments aimed at identifying substrate determinants, including the pentose ring, for binding and catalysis by E. coli DNA photolyase we tested several Pyr mean value of Pyr. We found that the enzyme has relative affinities for photodimers of T mean value of T greater than or equal to U mean value of T greater than U mean value of U much greater than C mean value of C and that the E-FADH2 form of the enzyme repairs these dimers at 366 nm with absolute quantum yields of 0.9 (T mean value of T), 0.8 (U mean value of T), 0.6 (U mean value of U), and 0.05 (C mean value of C). The enzyme also repairs an isolated thymine dimer and the synthetic substrate, 1,1'-trimethylene-bis (thymine) cyclobutane dimer. Unexpectedly, we found that this enzyme, previously thought to be specific for DNA, repairs uracil cyclobutane dimers in poly(rU). The affinity of photolyase for a uracil dimer in RNA is about 10(4)-fold lower than that for a U mean value of U in DNA; however, once bound, the enzyme repairs the photodimer with the same quantum yield whether the dimer is in ribonucleoside or deoxyribonucleoside form.
Kim
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Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill 27599.
Sancar
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D001482:Base Composition; D001665:Binding Sites; D002384:Catalysis; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008190:Lyases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010755:Organophosphates / Q000737:chemistry*; D010429:Pentoses / Q000737:chemistry*; D010940:Plant Proteins / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D013379:Substrate Specificity
D010755:Organophosphates; D010429:Pentoses; D010940:Plant Proteins; D011740:Pyrimidine Dimers; D012313:RNA; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase