A NOVEL DNA N-GLYCOSYLASE ACTIVITY OF ESCHERICHIA-COLI T4 ENDONUCLEASE-V THAT EXCISES 4,6-DIAMINO-5-FORMAMIDOPYRIMIDINE FROM DNA, A UV-RADIATION-INDUCED AND HYDROXYL RADICAL-INDUCED PRODUCT OF ADENINE

We report on a novel activity of T4 endonuclease V. This enzyme is wel l known to be specific for the excision of pyrimidine dimers from UV-i rradiated DNA. In this work, we show that T4 endonuclease V excises 4, 6-diamino-5-formamidopyrimidine from DNA. 4,6-Diamino-5-formamidopyrim idine is formed as a product of adenine in DNA upon action of hydroxyl radicals and upon UV-irradiation. DNA substrates were prepared by UV- or gamma-irradiation of DNA in aqueous solution. DNA substrates were i ncubated either with active T4 endonuclease V or with heat-inactivated T4 endonuclease V or without the enzyme. After incubation, DNA was pr ecipitated and supernatant fractions were separated. Supernatant fract ions after derivatization, and pellets after hydrolysis and derivatiza tion were analyzed by gas chromatography/isotopedilution mass spectrom etry. The results provide evidence for the excision of 4,6-diamino-5-f ormamidopyrimidine by T4 endonuclease V from both gamma-and UV-irradia ted DNA. Kinetics of excision were also determined. Fifteen other pyri midine- and purine-derived base lesions that were identified in DNA sa mples were not substrates for this enzyme. It was concluded that, in a ddition to its well known activity for pyrimidine photodimers, T4 endo nuclease V possesses an N-glycosylase activity for a major UV-radiatio n- and hydroxyl radical-induced monomeric product in DNA.