The Mechanism of Adenovirus‐DNA Synthesis in Isolated Nuclei

KB cells were productively infected with adenovirus type 5 and nuclei were isolated during the period of active viral DNA replication. The DNA synthesis in isolated nuclei was followed by studying the incorporation of deoxyribonucleoside triphosphates, including dBTP, which replaced dTTP completely. Sucrose gradient centrifugation of new DNA, synthesized during a 2‐h incubation, revealed the presence of three classes of viral DNA, one cosedimenting with mature viral DNA (31 S), one faster‐sedimenting class which contained replicating molecules and a slower‐sedimenting class which was not further investigated. Newly synthesized 31‐S DNA banded in CsCI within the range of light‐to‐hybrid DNA while less than 3% banded at higher densities. This suggests the absence of reinitiation of new rounds of DNA replication during the incubation period. The DNA synthesis was shown to proceed in a semiconservative fashion with a rate of at least 50% of the rate reported to occur in intact cells. By means of pulse labelling with [ 3 H]thymidine of DNA in intact cells followed by incubation of nuclei in the presence of dBTP it was found that a large fraction of the DNA strands involved in replication in intact cells was covalently bound to DNA synthesized in the nuclear system. This indicates that DNA synthesis in isolated nuclei continues at the replication fork that existed in the intact cells. During this process replicating molecules can be completed. Infection with adenovirus type 5 containing [ 32 P]DNA showed that parental DNA participated in the replication process. A covalent binding between parental and new DNA was not observed, neither in replicating DNA nor in the 31‐5 mature DNA. The latter observation showed the absence of extensive repair synthesis.