Light‐induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy

Abstract

Cyclobutane pyrimidine dimers (Pyr< >Pyr) and pyrimidine-pyrimidone (6-4) photoproducts are the predominant structural modifications resulting from exposure of DNA to ultraviolet light [1,2] 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 15 N-labelled enzyme and on experiments with D 2 O 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 lightdriven DNA repair process.Abbreviations DTT, dithiothreitol; FT-IR, Fourier transform infrared; MTHF, 5,10-methenyltetrahydrofolylpolyglutamate; Pyr<>Pyr, cyclobutane pyrimidine dimmer.

Fourier-Transform Infrared Study of the Photoactivation Process of Xenopus (6–4) Photolyase

“…Previous study suggested that the bands at 1489 (+), and 1397 (+) cm −1 originate from the C=N stretching vibration, and in-plane rocking mode for N 5 -H, respectively. 32,42 …”

Magnetic-field effect on the photoactivation reaction of Escherichia coli DNA photolyase

“…E. coli DNA photolyase contains two noncovalently bound cofactors, FAD and 5,10-methenyltetrahydrofolate (MTHF), which are involved in redox chemistry and light harvesting, respectively (2,5). A non-MTHFbinding mutant of E. coli DNA photolyase (E109A) (EcPL) (35) was used in the present studies to avoid sample inhomogeneity arising from the low binding constant of MTHF to the wild-type enzyme, and to prevent MTHF photodecomposition as a result of prolonged blue light illumination (36). Recombinantly expressed, catalytically competent EcPL is typically isolated with its FAD cofactor in the blue-semiquinone radical state, FADH • .…”

“…The expression and purification of EcPL are described elsewhere (35). Enzyme activity was monitored following the procedure developed by Jorns et al (47).…”

Light-driven DNA repair by photolyases

“…After DNA repair, the thymine pair has to flip back into the duplex DNA to form hydrogen bonds with the complementary adenines. This relaxation of the DNA backbone proceeds much more slowly than the repair of CPD lesions, as was shown by FTIR spectroscopy [68].…”