Abstract
We have purified DNA photolyase from the autotrophic anaerobic archaebacterium Methanobacterium thermoautotrophicum to near homogeneity by a two-column affinity chromatography. The purified enzyme has an Mr = 60,000 and shows near UV absorption peak at 440 nm and a fluorescence emission maximum at 462 nm indicating that it contains 8-hydroxy-5-deazaflavin (coenzyme F420) as an intrinsic chromophore. The photolyase binds with high specificity to thymine dimer in DNA with an equilibrium binding constant, KA = 1.4 x 10(9) M-1, and a dissociation rate constant, koff = 1.4 x 10(-4) s-1 (t1/2 = 43 min). Despite 6-fold higher affinity compared to the folate-containing Escherichia coli photolyase the two enzymes apparently contact the same phosphates around the thymine dimer: the phosphate immediately 5' and the three phosphates immediately 3' to the dimer on the damaged strand and the phosphate across from the dimer in the minor groove on the complementary strand. The absolute action spectrum of the Methanobacterium photolyase in the 400-500-nm region closely matches the absorption of the enzyme-bound F420. The quantum yield (phi) over this region is constant and is approximately 0.2. The value is measurably smaller than the quantum yields reported for other DNA photolyases.
Highlights
We have purified DNA photolyase from the auto- 440 nm
DNA photolyases (EC 4.1.99.3) convert light energy into chemical energy to repair pyrimidine dimers introduced into DNA by far UV (200-300 nm) radiation
UV Single Strand DNA-Agarose-For affinity chromatography of photolyase UV-irradiated single strand DNA-agarose was prepared according to the method of Arndt-Jovin et al (1975)
Summary
DNA photolyases (EC 4.1.99.3) convert light energy into chemical energy to repair pyrimidine dimers introduced into DNA by far UV (200-300 nm) radiation. Chemicals-Heparin-agarose (Type I), calf thymus DNA, Sepharose CL-2B-300, dimethylsulfate, and ethylnitrosourea were purchased from Sigma, and 5 M CNBr in acetonitrile was obtained from Aldrich. UV Single Strand DNA-Agarose-For affinity chromatography of photolyase UV-irradiated single strand DNA-agarose was prepared according to the method of Arndt-Jovin et al (1975). Sonicated calf thymus DNA (3.6 mg/ml) was irradiated with UV from a germicidal lamp for 1h a t a distance of 10 cm prior to coupling. The gel retardation assay was used with pure enzyme to study the dark reaction (binding) as well as to obtain the photolytic cross-section ( 6 4 ) of the photochemical reaction. This assay was conducted as described by Husainand.
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