The adenovirus DNA binding protein effects the kinetics of DNA replication by a mechanism distinct from NFI or Oct-1

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Adenovirus DNA polymerase (Ad pol) is a eukaryotictype DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increased K m (app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation.

mentioning

confidence: 60%

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

Brenkman¹,

Heideman²,

Truniger³

et al. 2001

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“…At higher dCTP concentrations all pTP⅐CAT products can be extended, indicating that it is an intermediate in replication (1). By using low dCTP concentrations sufficient amounts of the intermediate product accumulate to enable the study of both products in one reaction.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

King

Teertstra

Vliet

1997

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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...

“…Like the SSBs of these phages, AdDBP cooperatively binds to the displaced DNA strand. However, AdDBP has additional roles during adenovirus DNA replication, like the indirect stimulation of the initiation step by increasing the binding of NFI to the replication origin (48,49), and a direct stimulation by lowering the K m for the first dNTP (50). Taking together these data with the differences displayed by GA-1 SSB with respect to the SSBs of the related phages 29 and Nf, it is possible that GA-1 SSB might be implicated in additional roles to those common to the replication systems of the three phages.…”

Section: Visualization Of the Deletion Mutant Proteins By Electronmentioning

confidence: 99%

Importance of the N-terminal Region of the Phage GA-1 Single-stranded DNA-binding Protein for Its Self-interaction Ability and Functionality

Gascón¹,

Carrascosa²,

Villar³

et al. 2002

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages 29 and Nf. In this work, the selfinteraction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo crosslinking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of 29 and Nf. Three deletion mutant proteins have been characterized, ⌬N19, ⌬N26, and ⌬N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein ⌬N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins ⌬N26 and ⌬N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.Single-stranded DNA-binding proteins (SSBs) 1 contribute to DNA metabolism, playing essential roles in different processes such as DNA replication, repair, and recombination (1-3). SSBs bind single-stranded DNA (ssDNA) in a selective, cooperative, and non-sequence-specific way, protecting it from nuclease attack and preventing the formation of secondary structures on it. SSBs are ubiquitous. They have been isolated from bacteria and their phages, eukarya and their viruses, and archaea (1-6).Different oligomerization states have been reported for SSBs. Thus, monomeric (T4gp32 from bacteriophage T4 or AdDBP from adenovirus (5)), dimeric (SSBs from filamentous phages M13 (1) and Pf3 (7)), homotetrameric (EcoSSB from Escherichia coli (2, 8) or human mitochondrial SSB (9)), and heterotrimeric (hRPA, human RPA (3)) SSBs have been described. Important differences have been also reported mainly concerning the amino acid sequence and ssDNA binding properties of the proteins of this family. The function of the SSB is particularly important in the case of organisms that replicate their genetic material via a proteinpriming mechanism followed by strand displacement, as large amounts of ssDNA are generated in the process (10 -12). This is the case of adenovirus and of the 29 family of Bacillus phages. The genome of the latter consists of a linear dsDNA molecule of about 20 kb that contains a phage-encoded terminal protein (TP) covalently linked to each 5Ј end. Replication of the viral genome starts at either DNA end non-simultaneously by a protein-priming mechanism. After a sliding-back step (13), the viral DNA polymerase elongates the initiation product proceeding by strand displacement toward the other DNA end. The displaced strand is cooperatively bound by the viral...