Helix-destabilizing properties of the adenovirus DNA-binding protein

Zijderveld, D. C.; Vliet, Peter C. van der

doi:10.1128/jvi.68.2.1158-1164.1994

Cited by 32 publications

(16 citation statements)

References 36 publications

(31 reference statements)

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“…In the presence of DBP, the polymerase becomes highly processive (Lindenbaum et al ., 1986) and the sensitivity for inhibitors like (S)‐HPMPApp is increased (Mul et al ., 1989). Based on the fact that no helicase activity or ATP is required during replication and the observation that DBP can unwind double‐stranded molecules up to 200 bp, it was suggested that DBP is involved in unwinding of the template during replication (Zijderveld and van der Vliet, 1994). We show that DBP is only required for elongation on a double‐stranded template.…”

Section: Discussionmentioning

confidence: 99%

“…Strand displacement synthesis does not require a helicase and is ATP independent (Pronk et al ., 1994). Since DBP has been shown to have helix‐destabilizing properties (Monaghan et al ., 1994; Zijderveld and van der Vliet, 1994), it has been suggested that DBP is required to unwind the double‐stranded template. Finally, DBP enhances the renaturation of complementary displaced strands (Zijderveld et al ., 1993).…”

Section: Introductionmentioning

confidence: 99%

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Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis

et al. 1997

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In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double‐stranded template. Elongation is dependent on the adenovirus DNA‐binding protein (DBP) which has helix‐destabilizing properties. DBP binds cooperatively to single‐stranded DNA (ssDNA) in a non‐sequence‐specific manner. The crystal structure of DBP shows that the protein has a C‐terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C‐terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single‐stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double‐stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double‐stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP‐independent unwinding of the template during adenovirus DNA replication.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis

et al. 1997

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“…In addition, it increases the rate of synthesis and processivity of the viral DNA polymerase and modifies its sensitivity to nucleotide analogues (Lindenbaum et al, 1986;Mul et al, 1989). DBP also has helix destabilizing properties that could be important for DNA unwinding during chain elongation (Zijderveld and Van der Vliet, 1994). Ad DBP also enhances initiation of replication by lowering the Km of formation of a pTP-dCMP initiation complex (Mul and Van der Vliet, 1993) and by increasing the binding of NFI to its recognition site in the auxiliary origin (Cleat and Hay, 1989;Stuiver and Van der Vliet, 1990).…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding.

et al. 1994

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The adenovirus single‐stranded DNA binding protein (Ad DBP) is a multifunctional protein required, amongst other things, for DNA replication and transcription control. It binds to single‐ and double‐stranded DNA, as well as to RNA, in a sequence‐independent manner. Like other single‐stranded DNA binding proteins, it binds ssDNA, cooperatively. We report the crystal structure, at 2.6 A resolution, of the nucleic acid binding domain. This domain is active in DNA replication. The protein contains two zinc atoms in different, novel coordinations. The zinc atoms appear to be required for the stability of the protein fold rather than being involved in direct contacts with the DNA. The crystal structure shows that the protein contains a 17 amino acid C‐terminal extension which hooks onto a second molecule, thereby forming a protein chain. Deletion of this C‐terminal arm reduces cooperativity in DNA binding, suggesting a hook‐on model for cooperativity. Based on this structural work and mutant studies, we propose that DBP forms a protein core around which the single‐stranded DNA winds.

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“…Surprisingly, NFI dissociates from the complex at an early stage, as a direct result of a conformational change in Adpol induced by binding of deoxynucleoside triphosphates (5). Other events in the disassembly of initiation complex and the transition between initiation and elongation remain poorly understood, although DBP, which is essential for elongation, has been shown to have helix-destabilizing properties and to increase processivity of Adpol (20,23,49). Virus DNA replication is followed by transcription and translation of late proteins and assembly of virus particles.…”

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confidence: 99%

Domain organization of the adenovirus preterminal protein

Webster

Leith

Hay

1997

J Virol

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In adenovirus-infected cells, the virus-encoded preterminal protein and DNA polymerase form a heterodimer that is directly involved in initiation of DNA replication. Monoclonal antibodies were raised against preterminal protein, and epitopes recognized by the antibodies were identified by using synthetic peptides. Partial proteolysis of preterminal protein reveals that it has a tripartite structure, with the three domains being separated by two protease-sensitive areas, located at sites processed by adenovirus protease. These areas of protease sensitivity are probably surface-exposed loops, as they are the sites, along with the C-terminal region of preterminal protein, recognized by the monoclonal antibodies. Preterminal protein is protected from proteolytic cleavage when bound to adenovirus DNA polymerase, suggesting either multiple contact points between the proteins or a DNA polymerase-induced conformational change in preterminal protein. Two of the preterminal protein-specific antibodies induced dissociation of the preterminal protein-adenovirus DNA polymerase heterodimer and inhibited initiation of adenovirus DNA replication in vitro. Antibodies binding close to the primary processing sites of adenovirus protease inhibited DNA binding, consistent with UV cross-linking results which reveal that an N-terminal, protease-resistant domain of preterminal protein contacts DNA. Monoclonal antibodies recognizing epitopes within the C-terminal 60 amino acids of preterminal protein stimulate DNA binding, an effect mediated through a decrease in the dissociation rate constant. These results suggest that preterminal protein contains a large, noncontiguous surface required for interaction with DNA polymerase, an N-terminal DNA binding domain, and a C-terminal regulatory domain. by guest http://jvi.asm.org/ Downloaded fromlittle is known about NFI, Adpol, and the subject of this study, pTP. Previous attempts to identify functionally important domains in pTP have been confined to insertion/deletion mutagenesis (10-12, 27, 33, 34), with the general conclusions from such studies being that regions spanning the protein are required for DNA replication and virus viability. Here we describe the domain structure of pTP determined by a combination of approaches, including antibody accessibility experiments, partial proteolysis, and photochemical cross-linking. This information was used to identify regions of pTP participating in interactions with DNA and Adpol. MATERIALS AND METHODSPurification of pTP, Adpol, DBP, NFI, and NFIII. Adenovirus type 2 (Ad2) pTP was expressed in Spodoptera frugiperda sf9 cells by using recombinant baculoviruses as described previously (41). Cells were resuspended in ice-cold 50 mM HEPES-NaOH (pH 8)-5 mM KCl-0.5 mM MgCl 2 supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 g of pepstatin per ml, 1 g of E-64 per ml, and 1 g of pepstatin per ml), incubated on ice for 10 min, then disrupted by 15 strokes in a Dounce homogenizer using a type B pestle. Nuclei were collected by centri...

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Helix-destabilizing properties of the adenovirus DNA-binding protein

Cited by 32 publications

References 36 publications

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis

Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding.

Domain organization of the adenovirus preterminal protein

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