Biological inactivation of pBR322 plasmid DNA by enzyme- and radiation-induced single-strand damage under various conditions.
The influence of three different kinds of single-strand breaks (ssb) on the biological activity of plasmid DNA (pBR322) was studied. The single-strand breaks were produced either by gamma-irradiation (together with base and sugar damage) or by DNase I digestion which introduced ligatable ssb. Non-ligatable ssb--single-strand gaps of three nucleotides in length--were generated in the nicked DNA by exonuclease III treatment. The biological activity (N/N(o)) of this damaged DNA was assessed in vivo by transformation of E. coli (CMK) repair wild-type cells. The activity of the enzymes of E. coli was studied in vitro by incubation in a protein extract of E. coli making use of an in vitro assay introduced earlier, which makes it possible to distinguish between enzymatic degradation (dsb formation) and repair of damaged plasmid DNA. The biological activity (D37) of DNA with non-ligatable ssb, as determined by electrotransformation, was about 56% lower than that of DNA with ligatable ssb. The biological activity of enzymatically damaged DNA is greater in calcium-treated cells than in electroporated cells. It is proposed that this is due to a calcium-dependent inhibition of nucleases. In contrast to the enzymatically damaged DNA, with gamma-radiation-damaged DNA a calcium-dependent increase in survival was not observed. Therefore, calcium-dependent nucleases do not play a role in the repair of damage produced by gamma-irradiation. The enzyme activity data show that the single-strand damages are either converted into dsb or repaired. A comparison of the efficiency of dsb formation in the extract for two of the single-strand damages is presented. The efficiency depends on the kind of damage and on the presence of cofactors, especially ATP and dNTPs.
Ventur
Y
Y
Max-Planck Institut für Strahlenchemie, Mülheim a.d. Ruhr, Germany.
Schulte-Frohlinde
D
D
D000818:Animals; D002417:Cattle; D004249:DNA Damage; D004274:DNA, Recombinant / Q000378:metabolism* / Q000528:radiation effects*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids
D004274:DNA, Recombinant; D003850:Deoxyribonuclease I