Molecular architecture of adenovirus DNA polymerase and location of the protein primer.
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.
Brenkman
Arjan B
AB
University Medical Centre, Department of Physiological Chemistry and Centre for Biomedical Genetics, Utrecht, The Netherlands.
Breure
Elise C
EC
van der Vliet
Peter C
PC
D000256:Adenoviridae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D011498:Protein Precursors; D014764:Viral Proteins; C085476:pTP protein, adenovirus; D004256:DNA Polymerase I; D005092:Exonucleases