Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA

Bertin, John; Sunstrom, Noelle-Ann; Acheson, Nicholas H.

doi:10.1128/jvi.67.10.5766-5775.1993

Cited by 6 publications

(9 citation statements)

References 73 publications

(100 reference statements)

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“…Binding of large T antigen to sites A, B, and C facilitates binding to site 1/2, in the core replication origin, where hexamers presumably form to initiate unwinding and DNA replication. The presence of these auxiliary sites (1,15,57) near the origin may therefore allow initiation of DNA replication at substantially lower concentrations of large T antigen than would be possible in their absence.…”

Section: Discussionmentioning

confidence: 99%

“…(ii) Point mutants. Point mutations were introduced individually into the consensus G(A/G)GGC binding sequences in binding sites A, B, and C within plasmid pGEM-Modori, as previously described (1). Mutants were named to describe which sites were mutated (see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…5A); e.g., mAmB specifies a mutant in which the two G(A/G)GGC sequences in site A and the two G(A/G)GGC sequences in site B were mutated. Mutant A was named A1A2 by Bertin et al (1).…”

Section: Methodsmentioning

confidence: 99%

“…Sites A, B, and C are located between the core replication origin and the early transcription unit. These sites contain, respectively, two, two, and four target pentanucleotide sequences in polyomavirus strain A3 and its derivatives (1,9,43,53). Adjacent pentanucleotides are spaced approximately one turn of the DNA helix apart in each of these three sites, implying that large T antigen molecules bound to adjacent pentanucleotides are aligned on one side of the helix.…”

mentioning

confidence: 99%

“…What is the role of polyomavirus large T antigen binding sites A, B, and C in viral DNA replication? Although these sites are not absolutely required to direct large T antigenmediated DNA replication in vivo (1,34,57), their presence augments DNA replication in transfected plasmids (57) and is required for optimal virus replication in permissive mouse cells (1). Large T antigen bound more strongly to sites A, B, and C than to site 1/2 in the absence of ATP (7,17,42), but ATP strongly increased the affinity of large T antigen for DNAs containing site 1/2 (27), probably by stimulating the formation of hexamers.…”

mentioning

confidence: 99%

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Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Peng

Acheson

1998

J Virol

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Polyomavirus large T antigen binds to multiple 5′-G(A/G)GGC-3′ pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a “handover” mechanism mediated by these protein-protein contacts.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…5A); e.g., mAmB specifies a mutant in which the two G(A/G)GGC sequences in site A and the two G(A/G)GGC sequences in site B were mutated. Mutant A was named A1A2 by Bertin et al (1).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Peng

Acheson

1998

J Virol

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Enhanced binding to origin DNA at low pH enables easy detection of polyomavirus large T antigen by gel mobility shift assay of unfixed complexes

Peng

Acheson

1999

Journal of Virological Methods

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Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function

Shivakumar

Das²

1994

Journal of General Virology

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The polyomavirus enhancer is separated from the early RNA initiation sites by a 120 bp promoter region. To identify the core promoter elements, we introduced base-substitution mutations within the potential elements in the vicinity of the RNA initiation site. Three of these mutants, two with mutations within a putative nuclear factor-1 (NF-1) binding site and the other within the TATA box, exhibited reduced promoter activity by about threefold in the mouse NIH 3T3 cell line. The activity of the other three mutants was either little affected or remained unchanged. Mobility shift assays using specific competitors and antibodies against NF-1 demonstrated the binding of a protein of the NF-1 family at a site adjacent to the TATA box, suggesting a role for NF-1 binding in early promoter function. The effect of these mutations was also evaluated in undifferentiated mouse embryonal carcinoma (F9) cells in the presence of an additional mutation (F441) at nucleotide position 5233. This additional mutation creates a strong binding site for a transcription factor, TEF-1, and helps the virus to grow in this cell line. While the TATA box and the GC box mutants behaved qualitatively in a similar fashion, the NF-1 motif now played a minor role in F9 cells. Western blot experiments demonstrated low levels of NF-1 protein in this cell line. The NF-1 motif partially overlaps a T-antigen binding motif and this motif is not involved in T-antigen-mediated regulation of the early promoter. Our results suggest that a protein of the NF-1 family binds to the core promoter and is important for early transcription in vivo. We further demonstrate that undifferentiated F9 cells contain a very low level of NF-1 and the F441 mutant possibly follows a different mechanism for promoter function in these cells.

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Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA

Cited by 6 publications

References 73 publications

Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Enhanced binding to origin DNA at low pH enables easy detection of polyomavirus large T antigen by gel mobility shift assay of unfixed complexes

Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function

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