THE DNA POLYMERASES INVOLVED IN ADENOVIRUS DNA REPLICATION

Abstract

Studies on the DNA polymerases that may play a role in Adenovirus (Ad) DNA replication have been hampered by the lack of appropriate mutants in these host coded enzymes. However, a variety of biochemical methods that enable the study of DNA replication will be described in this thesis. In order to study the proteins involved in this process, a system capable of synthesizing Ad DNA in vitro has been utilized. In addition, a specific inhibitor of (alpha)-polymerase has been used to implicate a role for this mammalian enzyme in viral DNA replication. Aphidicolin, a tetracyclic diterpenoid antibiotic, inhibits (alpha)-polymerase activity without affecting either (beta) or (gamma)-polymerase. This drug neither breaks duplex DNA nor inhibits thymidine uptake in drug treated cells. In addition, the drug had no effect on RNA or protein synthesis. DNA synthesis was inhibited 50% by 3 x 10('-6)M aphidicolin when measured in the intact adenovirus infected HeLa cell and in an infected nuclear extract in vitro system. Similar dose response curves were obtained when testing the inhibitory effect of aphidicolin on (alpha)-polymerase activity on nicked salmon sperm DNA. These results suggested a possible role for (alpha)-polymerase in the replication of Ad DNA.;The in vitro system used in the laboratory has the ability to initiate DNA synthesis on adenoviral DNA that is covalently linked to a 5' terminal protein (DNA-PRO complex). The system is dependent upon the presence of two separate components; a soluble extract of uninfected HeLa cell nuclei and a 25-60% ammonium sulfate precipitate of adenovirus infected cytoplasm. Further investigation to define the requirements furnished by the uninfected nuclear extract have indicated that the active factors are present both in G(,1) and S phase nuclei of the HeLa cell cycle. The majority of the activity contributed by these various nuclear extracts from uninfected cells could be replaced with purified preparation of (beta)-polymerase. This reaction, (beta)-polymerase plus Ad infected cytoplasms, was also dependent upon addition of ATP and required the adenovirus DNA-PRO complex to yield full length Ad DNA products. The ability of (beta)-polymerase to take part in this reaction is presumably by complementing the abundant amount of (alpha)-polymerase found in the cytoplasm. Although these results indicated that (beta)-polymerase could participate in the in vitro synthesis of Ad DNA, it is not yet clear that this is indeed the case in vivo.;Chinese Hamster Ovary (CHO) cells have previously been thought to be non-permissive for Ad infection. The data in this thesis will show that CHO cells can be infected with Ad and support the synthesis of viral DNA. The yield of viral DNA was the same as that produced in Ad infected HeLa cells, but no virion was produced in CHO cells. Adenovirus DNA synthesis in extracts of CHO cells correlates well with studies in these cells in culture. Extracts from the cytoplasm of Ad infected CHO cells added to extracts of uninfected CHO nuclei synthesized full length viral DNA upon addition of adenovirus DNA-PRO complex. This replication system has the potential to probe host coded factors as well as viral factors involved in DNA synthesis. With the increasing number of conditionally lethal mutants recognized in CHO cells, it may be possible to use the in vitro Ad DNA replication system to determine how a particular mutation affects DNA replication.