Molecular architecture of adenovirus DNA polymerase and location of the protein primer

Abstract

Adenovirus (Ad) DNA polymerase (pol) belongs to the distinct subclass of the polalpha family of DNA pols that employs the precursor terminal protein (pTP) as primer. Ad pol forms a stable heterodimer with this primer, and together, they bind specifically to the core origin in order to start replication. After initiation of Ad replication, the resulting pTP-trinucleotide intermediate jumps back and pTP starts to dissociate. Compared to free Ad pol, the pTP-pol complex shows reduced polymerase and exonuclease activities, but the reason for this is not understood. Furthermore, the interaction domains between these proteins have not been defined and the contribution of each protein to origin binding is unclear. To address these questions, we used oligonucleotides with a translocation block and show here that pTP binds at the entrance of the primer binding groove of Ad pol, thereby explaining the decreased synthetic activities of the pTP-pol complex and providing insight into how pTP primes Ad replication. Employing an exonuclease-deficient mutant polymerase, we further show that the polymerase and exonuclease active sites of Ad pol are spatially distinct and that the exonuclease activity of Ad pol is located at the N-terminal part of the protein. In addition, by probing the distances between both active sites and the surface of Ad pol, we show that Ad pol binds a DNA region of 14 to 15 nucleotides. Based on these results, a model for binding of the pTP-pol complex at the origin of replication is proposed.

FIG. 1.

Distance between the exonuclease active site and the entrance of the primer binding groove. (A) Schematic representation of Ad pol based on the crystal structure of the replicating RB69 DNA pol complex (11). The lower part of the polymerase shows the palm domain containing the polymerase active center (P) and the thumb. The upper part of the polymerase shows the exonuclease domain with its active center (E). The N-terminal domain and the fingers have been left out for clarity. The gray area indicates the grooves where DNA can bind. The entrances of the primer and template binding grooves are depicted. 5′-Labeled D7bio is schematically presented as a solid line or as a broken line when is covered by streptavidin (S). The biotin group (Bio) is depicted as a box. (B) For Ad pol, exonucleolytic degradation was studied on 5′-labeled 20-mer D7bio, which contains a biotin group at position 7. Degradation was studied for the indicated times in the absence (lanes 1 to 6) or presence (lanes 7 to 12) of streptavidin. Arrows indicate accumulated degradation products and the position of the biotin group (bio). The measured distance is presented as a double-headed arrow. (C) For the pTP-pol complex, exonucleolytic degradation of the 5′-labeled 30-mer D7bio10 was studied in the absence (lanes 1 to 5) or presence (lanes 6 to 10) of streptavidin for the indicated times. Arrows indicate accumulated degradation products and the position of the biotin group. The measured distance is presented as a double-headed arrow.

FIG. 2.

Distance between polymerase active site and entrance of the template binding groove. (A) Schematic representation of the experiment. See the legend to Fig. 1A for details. (B) Elongation of Ad pol was studied on primer/template D20/Tbio5′ in the absence (lanes 2 to 6) or presence (lanes 7 to 11) of streptavidin for the indicated times. Lane 1 is a control elongation reaction performed on primer/template D20/T30. Arrows indicate accumulated products. The double-headed arrow depicts the measured distance between the entrance of the template binding groove and the polymerase active site. (C) Elongation of D20/Tbio5′ was studied on Ad pol and the pTP-pol complex in the absence (−) or presence (+) of streptavidin for 15 min at 37°C.

FIG. 3.

Characterization of the exonuclease-deficient mutant polymerase D422A. Mutant polymerase D422A was tested for exonuclease activity on 5′-labeled D20 and for polymerase activity on primer/template D20/T30 as described in Materials and Methods. (A) Exonucleolytic degradation was studied on D20 for wild-type (Wt) Ad pol and mutant polymerase D422A. The incubation times were as follows: 1 min (lanes 2 and 6), 5 min (lanes 3 and 7), 10 min (lanes 4 and 8), and 15 min (lanes 5 and 9). (B) To study the elongation of Wt Ad pol and mutant polymerase D422A, a DNA pol- and exonuclease-coupled assay on D20/T30 was performed in the presence of the following increasing concentrations of dNTPs: 25 nM (lanes 1 and 5), 125 nM (lanes 2 and 6), 625 nM (lanes 3 and 7), and 2,500 nM (lanes 4 and 8). Incubation was at 37°C for 10 min.

FIG. 4.

Distance between the polymerase active site and the entrance of the primer binding groove. (A) Schematic representation of the experiment. See the legend to Fig. 1A for details. (B) Partially degraded D20 was hybridized to template T30. The resulting primer/template mixture (lane 1) was elongated at 30°C for the indicated times in the absence (lanes 2 to 4) or presence (lanes 5 and 6) of streptavidin by the exonuclease-deficient mutant polymerase D422A. Arrows indicate the length of the elongation products.

FIG. 5.

Model for origin binding of the pTP-pol complex preceding replication initiation. The Ad origin DNA (thick lines) containing the core origin (nucleotides 9 to 18) are bound by Ad pol. pTP is complexed to Ad pol with its priming part depicted at the primer binding cleft (arrow) close to the polymerase active site (P) and the templating nucleotide for initiation (nucleotide 4 of the template strand). The exonuclease active site (E) has been indicated for clarity. The NH₂ domain represents the putative N-terminal domain of Ad pol that could bind to the core origin. See text for more details.