Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA

Abstract

We have carried out a series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli. In many respects, these effects differ greatly from those previously described for non-density-labeled (light) phage. Moreover, our results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication, strand displacement, and a considerable amount of DNA replication. UV irradiation of 5-bromodeoxyuridine-labeled phage results in single-stranded breaks in a linear, dose-dependent manner (1.3 to 1.5 breaks per genomic strand per lethal hit). This damage does not interfere with injection of the phage genome, but some of the UV-irradiated heavy phage DNA undergoes additional intracellular breakdown (also dose dependent). However, a minority (25%) of the injected parental DNA is protected, maintaining its preinjection size. This protected moiety is associated with a replicative complex of DNA and proteins, and is more efficiently replicated than is the parental DNA not so associated. Most of the progeny DNA is also found with the replicative complex. The 5-bromodeoxyuridine of heavy phage DNA is debrominated by UV irradiation, resulting in uracil which is removed by host uracil glycosylase. Unlike the simple gap-filling repair synthesis after infection with UV-irradiated light phage, the repair replication of UV-irradiated heavy phage is extensive as determined by density shift of the parental label in CsC1 gradients. The newly synthesized segments are covalently attached to the parental fragments. The repair replication takes place even in the presence of chloramphenicol, a protein synthesis inhibitor, suggesting it is host mediated. Furthermore, the extent of the repair replication is greater at higher doses of UV irradiation applied to the heavy phage. This abundant synthesis results ultimately in dispersion of the parental sequences as short stretches in the midst of long segments of newly synthesized progeny DNA. Together, the extensive replication and the resulting distribution pattern of parental sequences, without significant solubilization of parental label, are most consistent with a model of repair synthesis in which the leading strand displaces, rather than ligates to, the encountered 5' end.