The folate cofactor of Escherichia coli DNA photolyase acts catalytically.
Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
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Hamm-Alvarez
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Sancar
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Rajagopalan
D002384:Catalysis; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005492:Folic Acid / Q000378:metabolism*; D007700:Kinetics; D010777:Photochemistry; D011485:Protein Binding; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism
D011624:Pteroylpolyglutamic Acids; D013763:Tetrahydrofolates; D005492:Folic Acid; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004255:Deoxyribodipyrimidine Photo-Lyase