Identification of a DNA binding domain in simian virus 40 capsid proteins Vp2 and Vp3.

The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.

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Two Regions of Simian Virus 40 T Antigen Determine Cooperativity of Double-Hexamer Assembly on the Viral Origin of DNA Replication and Promote Hexamer Interactions during Bidirectional Origin DNA Unwinding

Journal of Virology ◽

10.1128/jvi.73.3.2201-2211.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2201-2211 ◽

Cited By ~ 52

Author(s):

Klaus Weisshart ◽

Poonam Taneja ◽

Andreas Jenne ◽

Utz Herbig ◽

Daniel T. Simmons ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Binding Domain ◽

Dna Unwinding ◽

Dna Binding Domain ◽

Wild Type ◽

Viral Origin

ABSTRACT Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

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Properties of the DNA-binding domain of the simian virus 40 large T antigen.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5525 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5525-5536 ◽

Cited By ~ 9

Author(s):

D McVey ◽

M Strauss ◽

Y Gluzman

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Dna Analysis ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Binding Domain ◽

Protein Domain ◽

Dna Binding Domain ◽

Single Stranded Dna

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.

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Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444911014302 ◽

2011 ◽

Vol 67 (6) ◽

pp. 560-567 ◽

Cited By ~ 5

Author(s):

Gretchen Meinke ◽

Paul Phelan ◽

Amélie Fradet-Turcotte ◽

Jacques Archambault ◽

Peter A. Bullock

Keyword(s):

Dna Binding ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Binding Domain ◽

Dna Binding Domain ◽

Large T Antigen ◽

Sv40 Large T Antigen ◽

Oligomeric Form

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The DNA-binding domain of simian virus 40 tumor antigen has multiple functions.

Journal of Virology ◽

10.1128/jvi.67.12.7608-7611.1993 ◽

1993 ◽

Vol 67 (12) ◽

pp. 7608-7611 ◽

Cited By ~ 37

Author(s):

K Wun-Kim ◽

R Upson ◽

W Young ◽

T Melendy ◽

B Stillman ◽

...

Keyword(s):

Dna Binding ◽

Tumor Antigen ◽

Simian Virus 40 ◽

Simian Virus ◽

Binding Domain ◽

Dna Binding Domain ◽

Multiple Functions

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Properties of the DNA-binding domain of the simian virus 40 large T antigen

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5525-5536.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5525-5536

Author(s):

D McVey ◽

M Strauss ◽

Y Gluzman

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Dna Analysis ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Binding Domain ◽

Protein Domain ◽

Dna Binding Domain ◽

Single Stranded Dna

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.

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