In vitro repair of ultraviolet-irradiated deoxyribonucleic acid : characterization of incision using the partially-purified UvrA, UvrB, and UvrCgene-products of Escherichia coli

Partially-purified UvrA, UvrB, and UvrC (UvrABC) gene products, prepared by column chromatography of extracts made from sucrose-plasmolyzed Escherichia coli, were used to help characterize in vitro the ability of E. coli to incise ultraviolet-irradiated DNA.The results of these experiments indicated that incision of irradiated PM2 DNA occurred with a 102 (20-40%) efficiency, but that the ability for incision to occur at every dimer was probably not limited by the amount of the gene products, DNA, ATP, Mg⁺, or KCl added to the reactions nor by the number of dimers Other experiments in the reactions, supported that the efficiency was not enhanced by the addition of the partially-purified UvrD gene product but was enhanced by incubation of the UvrABC gene products prior to the addition of the DNA. amount of incision was dependent upon the time the reactions were The incubated and linearly dependent upon the UV dose applied to the DNA. The data are consistent with incision occurring at a constant proportion of the damage in these reactions and the UvrABC gene products acting by a processive mechanism, The complete in vitro repair of irradiated bacteriophage T7 DNA using enzymes (the partially-purified UvrABC gene products and a crude extract) originating solely from its host was also investigated since this should be instructive relative to repair in E. coli in vitro, Repair was monitored with both physicochemical (restoration of mode molecular weight) and biological (enhanced ability of repaired DNA to form viable phage when encapsulated in vitro into phage heads) endpoints. The results of these experiments showed no significant difference in biological activity between DNA repaired in vitro or not repaired although incision occurred at as much as 70% of the dimers and although the molecular weight of the DNA was 90% restored in the reactions incubated with the UvrABC proteins and an extract from sucrose-plasmolyzed E. coli. These data support that the completeness of the in vitro repair of bacteriophage T7 DNA in these reactions may be limited in some step other than incision.