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

Abstract

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...

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

“…In this regard, it is informative to compare protein-primed DNA synthesis in hepadnaviruses to that in other viruses (such as the adenovirus and the phage 29) that also employ protein priming to initiate DNA synthesis. 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).…”

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

“…The transition from initiation to elongation in adenovirus DNA replication is characterized by a jumping-back mechanism, which recovers the terminal three nucleotides, resulting in DNA primer-template elongation and dissociation of pTP (3,4). To study elongation after protein priming, truncated replication reactions were performed in the presence of 50 nM dCTP and additionally 40 M dATP and 40 M dTTP but without dGTP.…”

“…Ad pol initiates replication opposite the fourth base of the template strand and synthesizes a pTP-CAT intermediate. For elongation to occur, this intermediate jumps back to be paired with template residues 1-3, after which pTP dissociates from Ad pol and elongation starts (3,4). Elongation occurs via a strand displacement mechanism that requires the viral DNAbinding protein (reviewed in Refs.…”

Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces

“…A terminal protein (TP) covalently bound to the 5Ј end of the phage DNA forms a heterodimer with the phage-encoded DNA polymerase, catalyzing covalent linkage of the TP to the first nucleotide of the nascent DNA strand and priming strand-displacing synthesis of full-length 29 DNA (Salas 1991;Salas et al 1995;Mendez et al 1997;Gonzales-Huici et al 2000). In eukaryotic cells, replication of adenoviruses, which also have protein bound covalently to 5Ј DNA termini, occurs by a fundamentally similar proteinprimed strand-displacement mechanism (van der Vliet 1995;Hay 1996;King et al 1997;de Jong and van der Vliet 1999;Liu et al 2000).…”