The (I/Y)XGG Motif of Adenovirus DNA Polymerase Affects Template DNA Binding and the Transition from Initiation to Elongation

Abstract

Adenovirus DNA polymerase (Ad pol) is a eukaryotictype DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. 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 substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increased K m (app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the 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 transition from initiation to elongation.

Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses

“…Due to the special nature of the protein primer, all protein-primed DNA syntheses studied so far require the polymerase to undergo some structural changes as it switches from an initiation mode, when the primer for DNA synthesis is a protein, to an elongation mode, when the primer for DNA synthesis is the newly formed DNA oligomer (18,21). However, the structural transition from the protein-primed initiation mode to the DNA-primed elongation mode has been shown to occur only after the synthesis of a DNA oligomer of at least several (from 3 to 9) nucleotides in the case of adenoviruses and the bacteriophages (5,18,21). In hepadnaviruses, the corresponding transition is likely the RT conformational change accompanying minus-strand template switch, also occurring after the synthesis of the 3-to 4-nucleotide DNA oligomer (i.e., the completion of protein priming) (27,32).…”

“…Clearly, the putative RT thumb subdomain is dispensable for the initiation of protein priming (step 3) but required for its completion (step 4). Following the completion of protein priming, the RT undergoes another major conformational change (RT 5 ), dissociates from the ε RNA and the RT-nascent minusstrand DNA complex, and then translocates to the 3Ј end of the pgRNA (minus-strand template switch) to continue DNA synthesis (elongation mode) (30,31).…”

Molecular Architecture of Adenovirus DNA Polymerase and Location of the Protein Primer

“…Wild-type Ad DNA pol was expressed from a baculovirus expression system and purified to near homogeneity as previously described (4). The exonuclease-deficient mutant polymerase D422A was constructed by performing site-directed mutagenesis on full-length Ad pol cDNA as described previously (4). The oligonucleotides for the PCR mutagenesis were 5Ј-ATCACCGGCTTTGCCGAGATCGTGCTC-3Ј and 5Ј-GAGCACGATCTCGGCAAAGCCGGTGAT-3Ј (changes marked in bold).…”

“…The presence of the desired mutation was confirmed by sequencing. Preparation of the recombinant baculoviruses, protein expression, and purification to near homogeneity were performed as described previously (4). The pTP-pol complex was purified as described previously (20).…”

“…Within this family, it is part of the subclass of protein-priming DNA pols (15). Mutational analysis of the polymerase domain has shown that, like other pol␣-like DNA pols, Ad pol is functionally conserved with the polymerase activity located at the C terminus (4,5,6,17,22,26,27). Biochemical analysis of Ad pol has shown that it replicates DNA in a processive manner but that it has a distributive 3Ј-5Ј exonuclease activity on single-stranded DNA, although removal of a mismatched nucleotide and subsequent switching to polymerization proceeds processively (18).…”