The (I/Y)XGG motif of adenovirus DNA polymerase affects template DNA binding and the transition from initiation to elongation

Adenovirus DNA polymerase (Ad poI) is a eukaryotic-type DNA polymerase invo lved in the catalysis of protein-primed initiation as well as DNA polymeriz ation. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-direc ted mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex D NA. Three mutant polymerases. required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double- stranded DNA. These observations suggest a local destabilization of DNA sub strate at the polymerase active site. In agreement with this, the mutant po lymerases showed reduced initiation activity and increased K-m(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while c apable of elongating on the primer-template DNA, failed to elongate after p rotein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and cease d completely after formation of the precursor terminal protein-trinucleotid e (pTP-CAT) initiation intermediate. Our results suggest that residues in t he conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the tran sition from initiation to elongation.