Cleavable Substrate Containing Molecular Beacons for the Quantification of DNA‐Photolyase Activity

Kundu, Lal Mohan; Burgdorf, Lars T.; Kleiner, Oliver; Batschauer, Alfred; Carell, Thomas

doi:10.1002/1439-7633(20021104)3:11<1053::aid-cbic1053>3.0.co;2-#

Cited by 38 publications

(42 citation statements)

References 57 publications

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“…Cyclobutane pyrimidine dimer repair by photolyase can be monitored indirectly based on UV absorbance changes (33), cleavable molecular beacons (34), and aminopurine fluorescence quenching (35), as well as gel electrophoresis (28,(36)(37)(38) and HPLC analysis (34); transformation assays also provide an in vivo assay (22). In our experiments, DNA repair is easily observed electrochemically as an increase in the redox signal of the flavin cofactor associated with the repair of the TϽϾT.…”

Section: Resultsmentioning

confidence: 97%

Electrically monitoring DNA repair by photolyase

DeRosa

Sancar

Barton³

2005

Proc. Natl. Acad. Sci. U.S.A.

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Cyclobutane pyrimidine dimers are the major DNA photoproducts produced upon exposure to UV radiation. If left unrepaired, these lesions can lead to replication errors, mutation, and cell death. Photolyase is a light-activated flavoenzyme that binds to pyrimidine dimers in DNA and repairs them in a reaction triggered by electron transfer from the photoexcited flavin cofactor to the dimer. Using gold electrodes modified with DNA duplexes containing a cyclobutane thymine dimer (T<>T), here we probe the electrochemistry of the flavin cofactor in Escherichia coli photolyase. Cyclic and square-wave voltammograms of photolyase deposited on these electrodes show a redox signal at 40 mV versus normal hydrogen electrode, consistent with electron transfer to and from the flavin in the DNA-bound protein. This signal is dramatically attenuated on surfaces where the -stacking of the DNA bases is perturbed by the presence of an abasic site below the T<>T, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically. Exposure of photolyase on T<>T-damaged DNA films to near-UV͞blue light leads to changes in the flavin signal consistent with repair, as confirmed by parallel HPLC experiments. These results demonstrate the exquisite sensitivity of DNA electrochemistry to perturbations in base pair stacking and the applicability of this chemistry to probe reactions of proteins with DNA.DNA charge transport ͉ DNA electrochemistry ͉ thymine dimers D NA-modified gold electrodes have proven to be invaluable tools in both the study and the application of DNAmediated charge transport (CT) chemistry (1-10). In these systems, the supramolecular self-assembly of thiol-modified DNA duplexes on a gold surface yields well defined monolayers that can serve as platforms for electrochemical assays of DNA CT. In these assays, the base pair stack is interrogated through the reduction of an intercalated reporter such as methylene blue (5, 7) or daunomycin (2, 8); the yield of reduced probe provides a measure of the efficiency of DNA CT. A wealth of experimental data has established that DNA CT is exquisitely sensitive to even subtle perturbations in the base pair -stack, including single base mismatches (3,8,9). Exploiting this dramatic effect, DNA-modified surfaces have been successfully used in the electrochemical detection of mismatches and base lesions (3, 9). Protein-DNA interactions can also be investigated by using these surfaces. Perturbations of the DNA base pair stack caused by protein binding through base flipping or kinking are clearly detected by using this methodology (10). Indeed, this technique can provide a sensitive probe of protein-DNA binding and reaction.The remarkable sensitivity of CT efficiency to DNA lesions and mismatches, as well as the observation that DNA-binding proteins can modulate long-range CT (10, 11), has provided an impetus for the evaluation of a physiological role for DNA CT. DNA-modified gold surfaces have recently been used in the study of DNA repair proteins posses...

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Section: Resultsmentioning

confidence: 97%

Electrically monitoring DNA repair by photolyase

DeRosa

Sancar

Barton³

2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Upon single-electron reduction, this dimer will induce a strand break as recently described by us. [12,35] Hairpin 19 is stable as long as the flavin molecule exists in the oxidized state. For the electron injection experiment, we prepared a solution of hairpin 19 in Tris-buffer (10 m Tris, c DNA ϭ 20 µ, pH ϭ 7.4) and added 50 µL of Figure 6.…”

Section: Excess Electron Transfer Studiesmentioning

confidence: 99%

Excess Electron Transfer in Flavin-Capped DNA-Hairpins

Behrens¹,

Ober²,

Carell³

2002

Eur. J. Org. Chem.

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“…Six-milliliter fractions were collected and dialysed against storage buffer (50 mM Tris, 50 mM NaCl, 10 mM DTT, 1 mM EDTA, 50% glycerol, pH 7.4). Table 1 Two types of oligonucleotides for preparing substrates 15-mer 5V-AGA GCA GTT GAC ACG-3V d(pT) 16 5V-TTT TTT TTT TTT TTT T-3V…”

Section: Purification Of His-tagged E Coli Photolyasementioning

confidence: 99%

“…Recently, reversed-phase high-performance liquid chromatography (RP-HPLC) method was employed to measure the activity of photolyase [16,17]. Based on their different hydrophobicity, the repaired DNA could be separated from the unrepaired DNA by reverse-phase column [18].…”

Section: Introductionmentioning

confidence: 99%