Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding

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Murine polyomavirus (Py) and simian virus 40 (SV40) encode homologous large T antigens (T Ags) and also have comparable sequence motifs in their core replication origins. While the ability of SV40 T Ag to produce specific distortions within the SV40 core replication origin (ori) in a nucleotide-dependent fashion has been well documented, little is known about related effects of Py T Ag on Py ori DNA. Therefore, we have examined viral origin DNA binding in the presence of nucleotide and the resulting structural changes induced by Py and SV40 T Ags by DNase I footprinting and KMnO 4 modification assays. The structural changes in the Py ori induced by Py T Ag included sites within both the A/T and early side of the core origin region, consistent with what has been shown for SV40. Interestingly, however, Py T Ag also produced sites of distortion within the center of the origin palindrome and at several sites within both the early and late regions that flank the core ori. Thus, Py T Ag produces a more extensive and substantially different pattern of KMnO 4 modification sites than does SV40 T Ag. We also observed that both T Ags incompletely protected and distorted the reciprocal ori region. Therefore, significant differences in the interactions of Py and SV40 T Ags with ori DNA may account for the failure of each T Ag to support replication of the reciprocal ori DNA in permissive cell extracts.

Section: Py and Sv40 T Antigens Cannot Support Replication Of Recipromentioning

confidence: 99%

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

Bhattacharyya

Lorimer

Prives

1995

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“…Both are required for the replication of their respective origins in cells (17) and in vitro (37,45,60,70). Polyomavirus large T antigen has DNA-binding (13,56), ATPase (9), and DNA helicase activ-ities (Wang and Prives, unpublished data) similar to those of SV40 large T antigen. Unlike the SV40 T antigen, polyomavirus large T antigen is not the principal transforming protein of polyomavirus; this activity resides with the middle T antigen (51, 64), a cytoplasmically localized protein which interacts with c-src at the plasma membrane (3,11).…”

mentioning

confidence: 99%

Binding of p53 and p105-RB is not sufficient for oncogenic transformation by a hybrid polyomavirus-simian virus 40 large T antigen

Manfredi

Prives

1990

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To identify regions on the large T antigens of simian virus 40 (SV40) and polyomavirus which are involved in oncogenic transformation, we constructed plasmids encoding hybrid polyomavirus-SV40 large T antigens. The hybrid T antigens were expressed in G418 sulfate-resistant pools of rat F2408 cells, and extracts of such pools were immunoprecipitated with an antibody against p53. Two hybrid T antigens containing SV40 amino acids 337 to 708 bound to p53, whereas another hybrid T antigen containing SV40 amino acids 412 to 708 did not. This suggests that a binding domain on SV40 large T antigen for p53 is contained within amino acids 337 to 708, with amino acids 337 to 411 playing an important role. One of the two hybrids that bound to p53 was chosen for further study. This T antigen contained SV40 large T antigen amino acids 336 to 708 joined to polyomavirus large T antigen amino acids 1 to 521 (PyT1 521-SVT336-708). Immunoprecipitation with antibodies directed against the product of the retinoblastoma susceptibility gene, p105-RB, showed that this hybrid bound p105-RB as well as p53. Pools expressing the hybrid PyTl-521-SVT336-708 did not grow in soft agar, nor did they form foci on confluent monolayers of nontransformed F2408 cells. The hybrid T antigen was expressed at levels comparable to those seen in retrovirus-infected F2408 cells expressing only SV40 large T antigen, which do show a transformed phenotype. Thus, this level of expression was sufficient for transformation by SV40 large T antigen but not for the hybrid large T antigen. These data, combined with genetic studies from other laboratories, suggest that complex formation with p53 and p105-RB is necessary but not sufficient for the oncogenic potential of papovavirus large T antigens. * Corresponding author. MATERIALS AND METHODS Plasmids, cells, and antibodies. pSVBam is plasmid pBR322 with the entire SV40 genome inserted into its BamHI site. pSVLT is the plasmid pMK 16 with the entire 5250

“…Previous DNA binding assays of polyomavirus or simian virus 40 large T antigen were carried out at various pH values between 7 and 8 (5,12,27,30,41,42,49,54). Although differences in binding to nonspecific (18,32,36) or specific (12) DNAs were noted, no systematic study of the variation of DNA binding with pH has been published.…”

Section: Resultsmentioning

confidence: 99%

“…Immunoprecipitation and DNase I protection assays showed that four distinct sites on polyomavirus DNA, denoted 1/2, A, B, and C, are bound by large T antigen in vitro (7,17,42). Site 1/2, which is situated within the core origin of DNA replication (23,26,28,34,43), contains four closely spaced consensus pentanucleotide sequences arranged symmetrically as two partly overlapping pairs on opposite DNA strands (8,9,41,49). Sites A, B, and C are located between the core replication origin and the early transcription unit.…”

mentioning

confidence: 99%

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

Peng

Acheson

1998

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.