Incision of ultraviolet-irradiated DNA by extracts of E. coli requires three different gene products

Abstract

IN most organisms pyrimidine dimers induced in DNA by ultraviolet light are removed by excision which is initiated by a repair-specific endonuclease that recognises the damage and makes a strand incision adjacent to the dimer^1–4. Characterisation of excision-defective mutants of Escherichia coli has shown that in this organism early steps of repair are controlled by the uvrA, uvrB and uvrC genes⁵. Although uvrA and uvrB mutants seem to be incision defective in vivo^6,7, it has not been possible to measure any difference in the amount of ultraviolet-endonuclease activity between crude extracts from mutants and wild-type cells^8,9. After partial purification of wild-type or uvrC mutant extracts, however, an ultraviolet endonuclease has been identified which is absent from uvrA and uvrB cells⁹ (in this communication termed the uvrAB endonuclease). The relevance of these results to whole cells is unclear, because recent experiments with permeable cells have shown that uvrA⁺B⁺-dependent strand incision requires adenosine-5′-triphosphate (ATP)^10–12, whereas the uvrAB endonuclease is independent of ATP². The aim of the present investigation was to observe ATP-dependent ultraviolet-endonuclease activity in a cell-free system. We report here the characterisation in crude extracts of an ATP-dependent ultraviolet-endonuclease activity from E. coli and conclude that the activity reflects that the enzyme is essential for repair in whole cells. The activity requires the complementary action of the uvrA⁺ uvrB⁺ and uvrC⁺ products and this has been utilised to establish in vitro assays for the individual products of these genes.