Substrate specificity of the Escherichia coli endonuclease III: excision of thymine- and cytosine-derived lesions in DNA produced by radiation-generated free radicals.

The excision of modified bases from DNA by Escherichia coli endonuclease III was investigated. Modified bases were produced in DNA by exposure of dilute buffered solutions of DNA to ionizing radiation under oxic or anoxic conditions. The technique of gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 16 pyrimidine- and purine-derived DNA lesions. DNA substrates were incubated either with the native enzyme or with the heat-inactivated enzyme. Subsequently, DNA was precipitated. Pellets were analyzed by GC/MS after hydrolysis and derivatization. Supernatant fractions were analyzed after derivatization without hydrolysis. The results provided unequivocal evidence for the excision by E. coli endonuclease III of a number of thymine- and cytosine-derived lesions from DNA. These were 5,6-dihydrothymine, 5-hydroxy-5-methylhydantoin, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil, alloxan, uracil glycol, and 5-hydroxy-6-hydrouracil. None of the purine-derived lesions was excised by endonuclease III. The present work extends the substrate specificity of E. coli endonuclease III to another thymine-derived and four cytosine-derived lesions. It is the first investigation of the substrate specificity of this repair enzyme in the context of a large number of pyrimidine- and purine-derived lesions in DNA.