The mechanism of replication of adenovirus DNA VI. Localization of the origins of the displacement synthesis

Abstract

Previous investigations have shown that replication of adenovirus type 5 (Ad5) proceeds via a displacement mechanism (see Levine et al., Curr. Topics Microbiol. Immunol. 6, for a review). This communication describes the localization of the origins of the displacement synthesis on the genome by restriction enzyme analysis of replicative intermediates synthesized during the first round of DNA replication. Replicative intermediates were isolated from KB cells infected with the temperature-sensitive mutant H5ts125, in which initiation of DNA replication is impaired. Infection was performed for 16 hr at the nonpermissive temperature (40°) during which no viral DNA synthesis occurred. Then the temperature was shifted to the permissive temperature (32°) and the cells were further incubated at this temperature. Replicative intermediates, labeled during various periods of time after shift-down, were isolated and subjected to restriction enzyme analysis. The highest specific radioactivity was found in restriction enzyme fragments which represent the two molecular ends. Preferentially only one of the two complementary strands at each molecular end was labeled. These results indicate that in the first round of replication the displacement synthesis may start at both molecular termini. The origin of the displacement synthesis of the viral r-strand is located at the molecular right-hand-end and synthesis of this strand proceeds from right to left, while the displacement origin of the viral l-strand is located at the molecular left-hand-end and synthesis of this strand proceeds in the opposite direction.