Adenovirus DNA polymerase: domain organisation and interaction with preterminal protein

Parker, Elizabeth J.; Botting, Catherine H.; Webster, Ailsa; Hay, Ronald T.

doi:10.1093/nar/26.5.1240

Cited by 24 publications

(18 citation statements)

References 47 publications

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“…Like other members of the Pol ␣ family, Adpol has a multidomain structure and noncontinuous regions of the polypeptide chains representing a large surface area are required for interaction with pTP (44,49). Analysis of the Adpol mutants described here allows the identification of residues that are likely to participate in the interactions with pTP and an assessment of their role in the initiation of DNA synthesis (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase

Liu

Naismith

Hay

2000

J Virol

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Human adenoviruses, of which there are over 40 different serotypes, are similar in morphology and genome organization. The genomes of human adenoviruses are linear doublestranded DNA (dsDNA) molecules of approximately 36,000 bp with inverted terminal repeats of about 100 bp, the precise size depending on the serotype. Located within the inverted terminal repeats are the cis-acting sequences which define the origin of DNA replication. Covalently attached to each 5' end of the genome DNA is a terminal protein (TP) which is likely to constitute an additional cis-acting component of the origin of DNA replication (reviewed in references 22 and 62).Replication of the adenovirus genome is catalyzed by adenovirus DNA polymerase (Adpol) via a protein-priming mechanism (reviewed in references 22 and 62) in which the adenovirus preterminal protein (pTP) acts as the protein primer. Adpol and pTP form a stable heterodimer, and following the binding of pTP-Adpol to the core origin of replication, DNA synthesis is initiated by Adpol catalyzing the addition of dCMP to the hydroxyl group of serine 580 of pTP. Initiation is enhanced by a virus-encoded single-stranded DNA (ssDNA) binding protein (DBP) and two cellular factors, nuclear factor I (NFI or CTF1) and nuclear factor III (NFIII or OCT1), are required for virus DNA replication (41,42,48,61). The presence of the adenovirus TP, which is covalently attached to the 5' end of the viral DNA, also stimulates initiation of DNA replication in vitro (46,47). Replication initiates opposite the GTA at positions 4 to 6 in the genome. After addition of dCMP, dAMP, and dTMP, the initiation product generated (pTP-CAT) jumps back to occupy positions 1 to 3 and then Adpol catalyzes the synthesis of the elongation product via a strand displacement mechanism (22).

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Section: Discussionmentioning

confidence: 99%

Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase

Liu

Naismith

Hay

2000

J Virol

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“…On the basis of the published sequence information, we examined various genes of the viral genome as possible target sequences for a quantitative PCR system. We focused on genes involved in DNA replication because these genes are known to be highly conserved (17)(18)(19). In alignments of published nucleotide sequences, only 1 region seemed suitable as a target for a generic probe.…”

Section: Discussionmentioning

confidence: 99%

Development of a PCR-Based Assay for Detection, Quantification, and Genotyping of Human Adenoviruses

Chmielewicz¹,

Nitsche²,

Schweiger³

et al. 2005

Clinical Chemistry

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“…Point and linker insertion mutants in this motif retained DNA elongation but had severely reduced initiation activity and lost the ability to bind the Ad core origin DNA, which is essential for Ad DNA replication (16). Furthermore, one of the four fragments of Ad pol obtained after partial digestion with endolys C contained these 250 amino acids, suggesting that it could fold as an independent domain (24). Alternatively, pTP could contribute to binding of the core origin.…”

Section: Discussionmentioning

confidence: 99%

“…Replication initiates via a protein-priming mechanism involving Ad pol and the primer pTP, which form the tight heterodimer pTP-pol. The precise interaction surface(s) between pTP and Ad pol has yet to be determined, although it has been suggested that the contacts extend over a large surface (24). Initiation of replication is catalyzed by Ad pol and can be stimulated by the two cellular transcription factors nuclear factor I and Oct-1, which function to recruit and position the pTP-pol complex to the origin of replication.…”

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confidence: 99%

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

Brenkman

Breure

Vliet

2002

J Virol

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Adenovirus (Ad) DNA polymerase (pol) belongs to the distinct subclass of the pol␣ 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.

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Adenovirus DNA polymerase: domain organisation and interaction with preterminal protein

Cited by 24 publications

References 47 publications

Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase

Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase

Development of a PCR-Based Assay for Detection, Quantification, and Genotyping of Human Adenoviruses

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

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