Identification of conserved residues contributing to the activities of adenovirus DNA polymerase

Adenovirus codes for a DNA polymerase that is a member of the DNA polymeras e a family and uses a protein primer for initiation of DNA synthesis. It co ntains motifs characteristic of a proofreading 3 ' -5 ' -exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase i n DNA replication, 22 site-directed mutations were introduced into the cons erved DNA polymerase motifs in the C-terminal region of adenovirus DNA poly merase and the mutant forms were expressed in insect cells using a baculovi rus expression system. Each mutant enzyme was tested for DNA binding activi ty, the ability to interact with pTP, DNA polymerase catalytic activity, an d the ability to participate in the initiation of adenovirus DNA replicatio n. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved resi dues in the various activities carried out by the protein. Using the functi onal data in this study and the previously published structure of the bacte riophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA pol ymerase function.