Endonuclease II of Escherichia coli: degradation of gammairradiated DNA.

Abstract

Irradiation of DNA in a nitrogen atmosphere with 60Co gamma-radiation produces at least two types of damage. The first type leads to single strand breaks in the DNA observed after exposure to alkali. This type of alkali-labile bond will be designated a spontaneous break. The second type of damage to DNA is an alteration which makes the DNA susceptible to phosphodiester bond hydrolysis by a 1600-fold purified preparation of endonuclease II of Escherichia coli and is designated an enzyme-sensitive site. This site is not alkali-labile. After irradiation, preincubation of the DNA either for days at 0 degrees or for 4 hr at 37 degrees increases both the spontaneous breaks and the enzyme sensitive sites. There is a greater increase of spontaneous breaks when the preincubation is in O2 compared to N2. The increase of enzyme sensitive sites due to the preincubation is not altered significantly by O2. The increase of spontaneous breaks during the preincubation is almost completely prevented by addition of either NaBH4 or NH2OH after the irradiation. The treatment can be before or after the preincubation. This effect indicates that these breaks are due to alkali-labile bonds possibly produced by depurination or depyrimidination reactions. That the spontaneous breaks are due primarily to alkali-labile bonds is supported by an experiment in which formamide gradients were used. Neither NaBH4 nor NH2OH has any effect on the enzyme sensitive sites. Addition of beta-mercaptoethanol (0.5 M) at the start of the preincubation prevents in part the appearance of both spontaneous breaks and enzyme-sensitive sites. It has no effect when added at the end of the preincubation. Catalase added before the preincubation has no effect on either type of damage. It is postulated that the spontaneous breaks occur because purine or pyrimidine radicals are formed (possibly hydroxyl radicals) which can then interact with oxygen to produce unstable intermediates. The intermediates then undergo either depurination or depyrimidination. The subsequent alkali catalyzed beta-elimination reaction of depurinated or depyriminidinated DNA is prevented by NaBH4 or NH2OH. An alternative hypothesis would involve damage to the sugar rather than to bases. The enzyme-sensitive sites represent another form of base damage which is not oxygen dependent. The chemical nature of either form of primary damage is not known.