Immunological characterization of the role of adenovirus terminal protein in viral DNA replication

Rijnders, A.W.M.; Bergen, B.G.M. Van; Vliet, Peter C. van der; Sussenbach, J.S.

doi:10.1016/0042-6822(83)90497-x

Cited by 7 publications

(7 citation statements)

References 13 publications

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“…However, these antibodies were not well characterized, and the inhibition occurred also in uninfected cells (27). Although we cannot completely rule out a role for pTP in vivo, for instance related to its property to attach to the nuclear matrix (28), our experiments make a role in vitro very unlikely.…”

Section: Dissociation Of the Ptp⅐pol Complex Occurs At Or Aftermentioning

confidence: 68%

Dissociation of the Protein Primer and DNA Polymerase after Initiation of Adenovirus DNA Replication

King

Teertstra

Vliet

1997

Journal of Biological Chemistry

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Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP⅐trinucleotide complex (pTP⅐CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP⅐pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP⅐C and pTP⅐CA were still bound to the polymerase, after the formation of pTP⅐CAT 60% of the pTP⅐pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and in K m for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA . . . instead of the natural GTAGTA . . . ) we show that G5 rather than G3 is used to start, leading to a pTP⅐tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two.The replication of the linear 36-kilobase pair adenovirus (Ad) 1 DNA initiates at two origins located at either molecular end employing a protein-priming mechanism. Initiation requires at least two viral proteins, the DNA polymerase (pol) and the precursor of the terminal protein (pTP), which acts as the primer and is linked covalently to the first nucleotide, a dCMP molecule. Initiation can be stimulated by the cellular transcription factors NFI and Oct-1 which guide the pTP⅐pol complex to the core origin employing their DNA binding domains. This leads to stabilization of the preinitiation complex and increases the number of initiation events by enhancing the V max of the reaction. In addition, the virus-coded singlestranded DNA-binding protein stimulates initiation by lowering the K m for dCTP (1).The virus uses a jumping back mechanism for initiation. At position 4 from the 3Ј-end of the template a pTP⅐CAT intermediate is synthesized which jumps back to be paired to the template residues 1-3 before elongation starts (2). This jumping or sliding-back mechanism was first described for 29 (3) and appears to be universal for protein-priming replication systems (4 -6), enabling errors made during initiation and small deletions to be restored. Subsequent elongation concomitant with the displacement of the non-template strand further requires the Ad DNA-binding protei...

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Section: Dissociation Of the Ptp⅐pol Complex Occurs At Or Aftermentioning

confidence: 68%

Dissociation of the Protein Primer and DNA Polymerase after Initiation of Adenovirus DNA Replication

King

Teertstra

Vliet

1997

Journal of Biological Chemistry

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“…Within this region, base pairs 9-18, which are perfectly conserved in all human adenovirus DNAs that have been sequenced, are essential (40, 107, 25 1, 287 , 301, 302, 32 1). Rijnders et al (255) reported that pTP-Ad Pol from Ad 5 binds specifically to restriction fragments containing the region of 9-22 in t h e Ad 2 DNA, equivalent to 9-18 in Ad 5, and on this basis h ave suggested t h at this sequence may play a role in the bind ing of pTP-Ad Pol to the origin. Though point mutations at position 4 from the terminus have no effect on either initiation or elongation, the nucleotides between 1 and 9 may serve as a spacer between the terminus and the conserved IO-base-pair core, since a one-base-pair deletion, four bases removed from the core sequence, inhibits formation of the pTP-dCMP complex (40,107).…”

Section: Dna Sequence Requirements For In Vitro Replicationmentioning

confidence: 99%

Eukaryotic Dna Replication

Campbell¹

1986

Annu. Rev. Biochem.

197

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“…Similar results obtained with deletion mutant pTP-CA32 and mutant pTP-A(235-243) suggest a function for pTP in Ad DNA replication after formation of the pTP-dCMP initiation complex. This might simply be related to the dissociation of Ad pol from the initiation complex or may point at a direct involvement of pTP in chain elongation, as suggested earlier on the basis of immunological studies (40).…”

Section: Discussionmentioning

confidence: 69%

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis

Roovers

Lee

Wees

et al. 1993

J Virol

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A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late genes in vitro and reintroduction of the mutated genes into the Ad genome (17,41). In our studies, a 12-bp synthetic oligonucleotide was inserted into multiple restriction sites 265 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from supernatant was used as virus stock for all further experiments. Titers of wt and mutant virus stocks were measured by plaque assay on tk-143 cells.

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Immunological characterization of the role of adenovirus terminal protein in viral DNA replication

Cited by 7 publications

References 13 publications

Dissociation of the Protein Primer and DNA Polymerase after Initiation of Adenovirus DNA Replication

Dissociation of the Protein Primer and DNA Polymerase after Initiation of Adenovirus DNA Replication

Eukaryotic Dna Replication

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis

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