DNA base damage induced by ionizing radiation recognized by Escherichia coli UvrABC nuclease but not Nth or Fpg...

Ionizing radiation and other free radical-generating systems induce a great variety of oxidative damage to DNA bases. The major known lesions are repaired by two well-characterized DNA glycosylases of Escherichia coli, endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), which have associated AP lyase activities. To detect and characterize potentially harmful oxidative base DNA lesions that may be repaired by alternative means, we exposed plasmid DNA to low doses of gamma-rays and removed the major base lesions by treatment with Nth and Fpg proteins. The closed circular DNA remaining after these treatments was used as a substrate of the UvrABC endonuclease complex from E. coli and as a template in a DNA polymerase arrest assay in vitro. The circular DNA contained lesions that were recognized and incised by the UvrABC nuclease and also lesions that blocked DNA polymerization in vitro. The blocking lesions were more abundant in DNA irradiated under nitrogen than under air and occurred mainly at tandem guanines; however, they were also frequent at tandem adenines and tandem cytosines.