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Electrically monitoring DNA repair by photolyase

DeRosa, Maria C. and Sancar, Aziz and Barton, Jacqueline K. (2005) Electrically monitoring DNA repair by photolyase. Proceedings of the National Academy of Sciences of the United States of America, 102 (31). pp. 10788-10792. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:DERpnas05

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Abstract

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 pi-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-UV/blue 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.


Item Type:Article
Additional Information:Copyright © 2005 by the National Academy of Sciences. Edited by Nicholas J. Turro, Columbia University, New York, NY, and accepted June 13, 2005 (received for review April 28, 2005). This paper was submitted directly (Track II) to the PNAS office. We thank Eric Han and Carrie Stentz for help with protein expression and purification. This work was supported by National Institutes of Health Grants GM61077 (to J.K.B.) and GM31082 (to A.S.) and a Natural Science and Engineering Research Council of Canada Postdoctoral Fellowship (to M.C.D.).
Subject Keywords:DNA charge transport, DNA electrochemistry, thymine dimers, escherichia-coli, electron-transfer, crystal-structure, charge-transport, thymine dimmers, protein, electrochemistry, identification, substrate, surfaces
Record Number:CaltechAUTHORS:DERpnas05
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:DERpnas05
Alternative URL:http://dx.doi.org/10.1073/pnas.0503527102
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:969
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Nov 2005
Last Modified:26 Dec 2012 08:42

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