Abstract

Mutants in highly conserved region I (YGDTDS) of the adenovirus serotyope 2 DNA polymerase (Ad Pol) have been shown previously to be defective in assays for initiation and elongation of adenovirus DNA replication in vitro. A selected subset of these mutants was characterized in a number of assays to determine in more detail the nature of the defect that they cause in Ad Pol. The single amino acid substitution in this sequence motif had no detectable effect on binding either to factors required for viral DNA replication or to Ad DNA origin. However, in the deletion mutant mimicking a similar sequence found in the Klenow fragment and in RNA polymerases, binding to Ad DNA origins was reduced. When the nucleotide and template specificity of partially purified mutant Ad Pol proteins was checked there were no significant differences between mutant and wild-type Ad Pols in RNA polymerase assays both on DNA templates and on RNA templates. In reverse transcription assays, both wild type and mutant Ad Pol were inactive, with the exception of a Gly to Met replacement mutant which mimicked the sequence found in reverse transcriptase; this mutant showed low but reproducible levels of reverse transcriptase activity when compared to wt Ad Pol. Taken together, these results suggest that region I may influence template specificity during DNA synthesis, although the single replacement is not sufficient to convert Ad Pol to a reverse transcriptase or an RNA polymerase. Region I probably acts independently of other regions of Ad Pol that are responsible for DNA binding, since mutations in this sequence did not significantly alter adenovirus DNA origin interactions in gel shift assays.

• Keywords: Adenovirus; DNA polymerare; nucleotide specificity; recombinant vaccinia virus; region I