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Nucleic Acids Research, 1987, Vol. 15, No. 3 1227-1243
© 1987

Articles

Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts

Gary M. Myles, Bennett Van Houten and Aziz Sancar

University of North Carolina, School of Medicine, Department of Biochemistry 231H Chapel Hill, NC 27514, USA

Received October 14, 1986. Revised December 18, 1986. Accepted December 18, 1986.

ABC excinuclease of Escherichia coli removes 6–4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from N. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6–4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyriaidine dimers (but not 6–4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.


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