Light-induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy.
Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH- chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH- form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH- form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.
Schleicher
Erik
E
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Germany.
Hessling
Benedikt
B
Illarionova
Viktoria
V
Bacher
Adelbert
A
Weber
Stefan
S
Richter
Gerald
G
Gerwert
Klaus
K
D001412:Bacillus subtilis / Q000235:genetics; D002384:Catalysis / Q000528:radiation effects; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000528:radiation effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008027:Light; D009154:Mutation / Q000235:genetics; D010777:Photochemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013941:Thymine / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects
D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; C001943:5-fluorouridine; C032418:5,11-methenyltetrahydrohomofolate; D005492:Folic Acid; D004255:Deoxyribodipyrimidine Photo-Lyase; D013941:Thymine; D014529:Uridine