SUBSTRATE-SPECIFICITY OF THE ESCHERICHIA-COLI ENDONUCLEASE-III - EXCISION OF THYMINE-DERIVED AND CYTOSINE-DERIVED LESIONS IN DNA PRODUCED BY RADIATION-GENERATED FREE-RADICALS

The excision of modified bases from DNA by Escherichia coli endonuclea se III was investigated. Modified bases were produced in DNA by exposu re of dilute buffered solutions of DNA to ionizing radiation under oxi c or anoxic conditions. The technique of gas chromatography/mass spect rometry (GC/MS) was used to identify and quantify 16 pyrimidine- and p urine-derived DNA lesions. DNA substrates were incubated either with t he native enzyme or with the heat-inactivated enzyme. Subsequently, DN A was precipitated. Pellets were analyzed by GC/MS after hydrolysis an d derivatization. Supernatant fractions were analyzed after derivatiza tion 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-hy droxy-5-methylhydantoin, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6 -dihydrouracil, alloxan, uracil glycol, and 5-hydroxy-6-hydrouracil. N one of the purine-derived lesions was excised by endonuclease III. The present work extends the substrate specificity of E. coli endonucleas e III to another thymine-derived and four cytosine-derived lesions. It is the first investigation of the substrate specificity of this repai r enzyme in the context of a large number of pyrimidine- and purine-de rived lesions in DNA.