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

H R Warner, L M Christensen, M L Persson
1981 Journal of Virology  
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 JV 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
more » ... ies 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 460C. Our results indicated that both of these enzyme activities are contained in the same enzyme molecule and, probably, in the same polypeptide. Recently, it has become clear that the incision of lightly irradiated DNA by the UV-specific endonuclease activity induced by bacteriophage T4 occurs in two steps (2, 14, 16, 20) . The first step is cleavage of the glycosylic bond between the pyrimidine and the deoxyribose in the 5' half of the pyrimidine dimer to produce an apyrimidinic (AP) site (Fig. 1) . The second step is nicking of the dimer-containing strand on the 3' side of the AP site generated by the glycosylase. The product of the denV gene of T4 is essential for induction of this UV endonuclease activity (4), and Seawell et al. (16) have demonstrated that the denV gene codes for the glycosylase activity. However, the data of these workers did not show whether the denV gene product also contains the AP endonuclease activity, and, in fact, Seawell et al. stated that the simplest interpretation of their data is that the den V gene codes for the t Paper 11,542 from the University of Minnesota Agricultural Experiment Station. pyrimidine dimer-DNA glycosylase but not for the AP endonuclease. In this paper we present several kinds of evidence suggesting that the glycosylase and the AP endonuclease activities both reside in the same protein and, probably, in the same polypeptide and that this polypeptide is the product of the denV gene. MATERIALS AND METHODS Bacteria and bacteriophage. Bacteriophage T4amN82 contains an amber mutation in gene 44 and is not able to synthesize phage DNA in the nonpermissive host Escherichia coli B/5 (22). We used this mutant because it overproduces early enzymes (22), and the UV endonuclease was thought to be an early enzyme (5). The amN82 mutation also prevented premature lysis of infected cells during harvesting. The
doi:10.1128/jvi.40.1.204-210.1981 fatcat:pthmjtyrlveitl7uccx3iqh7ze