Murine polyomavirus and simian virus 40 large T antigens produce different structural alterations in viral origin DNA

Bhattacharyya, Subarna; Lorimer, Heather E.; Prives, Carol

doi:10.1128/jvi.69.12.7579-7585.1995

Cited by 15 publications

(8 citation statements)

References 69 publications

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“…Taken together, our data and those of Bhattacharyya et al (2) suggest that ATP, or AMPPNP, induces the formation of a complex of large T antigen that covers the central part of site 1/2 over a single turn of the DNA helix. In contrast, simian virus 40 large T antigen protects a larger region of the homologous site II against DNase I digestion (5,38), and there are no KMnO 4 -sensitive sites generated at the equivalent positions in site II (2). Simian virus 40 large T antigen has been shown to form double hexamers at site II via a cooperative process directed by the two halves of site II (38).…”

Section: Discussionsupporting

confidence: 85%

“…In addition, on binding of large T antigen, there is increased DNase I sensitivity at positions located between sites A, B, and C ( Fig. 6) (2,30), consistent with the bending or distortion of the DNA helix that would be necessary to bring the various large T antigen molecules into contact with each other. However, we do not claim to know either the exact path of the DNA through this multiprotein complex or how stable such a complex would be in the cell.…”

Section: Discussionmentioning

confidence: 63%

“…Since polyomavirus large T antigen purified from P. pastoris had not been previously characterized, we asked whether an increase in DNA binding at a pH below 7 was peculiar to this source. We therefore purified polyomavirus large T antigen made in insect cells by a recombinant baculovirus (45) which has been used extensively in other studies (2,27,29,55,56) and measured its DNA binding activity as a function of pH; the results (not shown) were similar to those shown in Fig. 1A.…”

Section: Resultsmentioning

confidence: 68%

“…Polyomavirus large T antigen initiates DNA unwinding and replication via elaborate interactions with the viral replication origin (2). Specific DNA binding by this 785-amino-acid protein is mediated by a domain that lies between amino acids 282 and 398, as defined by using deletion mutants (52).…”

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: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 68%

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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“…A cellfree replication system was developed in which Py T Ag mediates replication of DNA containing the Py replication origin (Py ori-DNA) in murine cell extracts (59,65). Py T Ag binding to the Py origin palindrome is stimulated by nucleotides (47), and in the presence of nucleotides it can also form hexamers (85) and melt sites within the origin (4,43). Additionally, Py T Ag can unwind origin DNA and display DNA helicase activity (71,84).…”

mentioning

confidence: 99%

The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication

Reynisdóttir

Bhattacharyya

Zhang

et al. 1999

J Virol

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The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G1 to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (dl141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.

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Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein

Krause

Rückert

Lurz

et al. 1997

Journal of Molecular Biology

115

153

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Murine polyomavirus and simian virus 40 large T antigens produce different structural alterations in viral origin DNA

Cited by 15 publications

References 69 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

The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication

Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein

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