Evidence that the UV endonuclease activity induced by bacteriophage T4 contains both pyrimidine dimer-DNA glycosylase and apyrimidinic/apurinic endonuclease activities in the enzyme molecule

Abstract

We performed experiments to determine whether the phage T4-induced UV endonuclease activity is a single protein containing both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities. The UV endonuclease activity is induced by the denV gene and codes for the glycosylase activity. We obtained several kinds of evidence that the protein containing the glycosylase activity also contains the apyrimidinic endonuclease activity. After chromatography on DEAE-cellulose, the two activities copurified during phosphocellulose chromatography and Sephadex G-100 chromatography, with a constant ratio of activities across the activity peaks. On Sephadex G-100 columns the molecular weights of the two activities agreed within 2,500 or less. When an extract of cells infected with the T4 V1 mutant was purified in exactly the same way as an extract of cells infected with T4 V1+, neither glycosylase nor apyrimidinic endonuclease activity was detected in the normal elution position of the T4 UV endonuclease activity. The glycosylase and apyrimidinic endonuclease activities were induced with similar kinetics, which were characteristic of immediate early rather than delayed early enzymes. This correlated well with the presumed major role of these activities in repairing thymine dimers in parental DNA before DNA replication begins. Finally, glycosylase and apyrimidinic endonuclease activities were lost in parallel during incubation of the enzyme at 46 degree C. Our results indicated that both of these enzyme activities are contained in the same enzyme molecule and, probably, in the same polypeptide.