Nuclease for DNA apurinic sites may be involved in the maintenance of DNA in normal cells.

Abstract

Verly and Paquette1,2 demonstrated an enzyme in Escherichia coli which produces single strand breaks in DNA near apurinic sites. The enzyme was purified from the bacterial crude extract by the method used by Friedberg et al.3 for the preparation of endonuclease II. After elimination of the nucleic acids by treatment with streptomycin sulphate, the proteins were fractionated with ammonium sulphate; the enzyme precipitated at between 45 and 80% saturation in ammonium sulphate and was subsequently chromatographed on DEAE-cellulose and phosphocellulose4,5. The purified enzyme was tested on DNAs labelled with 3H-thymidine, normal DNA, DNA alkylated with methyl methanesulphonate containing 550 methyl groups per 106 molecular weight and depurinated DNA obtained by heating the alkylated DNA at 50° C during 6 h and containing about 160 apurinic sites per 106 molecular weight. With each substrate, the perchloric acid-soluble radioactivity was measured after different times of incubation. The acid-soluble radioactivity in controls without enzyme did not increase during incubation at 37° C. Figure 1 shows that the purified enzyme had no action on normal DNA and that its action on alkylated DNA was greatly enhanced when some of the alkylated sites were replaced by apurinic sites. The absence of action of the purified enzyme on normal DNA was further demonstrated with a very sensitive technique. The sedimentation profile of labelled DNA from T7 phage in a sucrose gradient after denaturation with NaOH was unaffected by incubation with the enzyme5. The purified enzyme is also without action on single-stranded DNA. It is known that alkylated DNA spontaneously loses alkylated purines6 and that the apurinic sites are quickly hydrolysed in an alkaline solution7,8. Figure 2 shows that the action of the purified bacterial enzyme on alkylated DNA is restricted to the apurinic sites resulting from the depurination process. The acid-soluble radioactivity was the same whether the labelled alkylated DNA was incubated with or without enzyme and then treated by NaOH; enzyme and NaOH thus cut the DNA strands at the same sites, that is, at the apurinic sites. We conclude that the purified bacterial enzyme has no action on normal DNA strands or on alkylated sites; it is entirely specific for apurinic sites.