Publications

• Direct observation of thymine dimer repair in DNA by photolyase.

  Kao Y.T., Saxena C., Wang L., Sancar A., Zhong D.

  Photolyase uses light energy to split UV-induced cyclobutane dimers in damaged DNA, but its molecular mechanism has never been directly revealed. Here, we report the direct mapping of catalytic processes through femtosecond synchronization of the enzymatic dynamics with the repair function. We obs ... >> More

  Photolyase uses light energy to split UV-induced cyclobutane dimers in damaged DNA, but its molecular mechanism has never been directly revealed. Here, we report the direct mapping of catalytic processes through femtosecond synchronization of the enzymatic dynamics with the repair function. We observed direct electron transfer from the excited flavin cofactor to the dimer in 170 ps and back electron transfer from the repaired thymines in 560 ps. Both reactions are strongly modulated by active-site solvation to achieve maximum repair efficiency. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site, with no net change in the redox state of the flavin cofactor. << Less

  Proc Natl Acad Sci U S A 102:16128-16132(2005) [PubMed] [EuropePMC]

• Studies on a DNA photoreactivating enzyme from Streptomyces griseus. II. Purification of the enzyme.

  Eker A.P., Fichtinger-Schepman A.M.

  A procedure is described for the isolation of a DNA photoreactivating enzyme from Streptomyces griseus. Application of chromatography on spherosil, ultraviolet-irradiated DNA-cellulose, DEAE-cellulose and single stranded-DNA-agarose resulted in a 22 000-fold purification with 46 percent recovery o ... >> More

  A procedure is described for the isolation of a DNA photoreactivating enzyme from Streptomyces griseus. Application of chromatography on spherosil, ultraviolet-irradiated DNA-cellulose, DEAE-cellulose and single stranded-DNA-agarose resulted in a 22 000-fold purification with 46 percent recovery on the initial activity. According to polyacrylamide gel electrophoresis the final preparation was virtually homogeneous. The absorption spectrum of the enzyme exhibited a marked absorption band in the 400-460 nm region in addition to protein absorption. << Less

  Biochim Biophys Acta 378:54-63(1975) [PubMed] [EuropePMC]

• Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex.

  Sancar G.B., Smith F.W., Reid R., Payne G., Levy M., Sancar A.

  Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integri ... >> More

  Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding. << Less

  J Biol Chem 262:478-485(1987) [PubMed] [EuropePMC]