Peter Tegtmeyer

p53 domains: structure, oligomerization, and transformation.

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

p53 oligomerization and DNA looping are linked with transcriptional activation.

We examined the role of p53 oligomerization in DNA binding and in transactivation. By conventional electron microscopy (EM) and scanning transmission EM, we find that wild‐type tetramers contact 18‐20 bp at single or tandem 19 bp consensus sequences and also stack in apparent register, tetramer on top of tetramer. Stacked tetramers link separated DNA binding sites with DNA loops. Interestingly, the p53(1‐320) segment, which lacks the C‐terminal tetramerization domain, binds DNA consensus sites as stacked oligomers. Although the truncated protein binds DNA with reduced efficiency, it nevertheless induces DNA looping by self‐association. p53, therefore, has a C‐terminal tetramerization domain that enhances DNA binding and a non‐tetrameric oligomerization domain that stacks p53 at consensus sites and loops separated consensus sites via protein‐protein interactions. Using model promoters, we demonstrate that wild‐type and tetramerization‐deficient p53s activate transcription well when tandem consensus sites are proximal to TATA sequences and poorly when tandem sites are distal. In the presence of proximal sites, however, stimulation by distal sites increases 25‐fold. Tetramerization and stacking of tetramers, therefore, provide dual mechanisms to augment the number of p53 molecules available for activation through p53 response elements. DNA looping between separated response elements further increases the concentration of local p53 by translocating distally bound protein to the promoter.

Reciprocal interference between the sequence-specific core and nonspecific C-terminal DNA binding domains of p53: implications for regulation.

The tumor suppressor p53 has two DNA binding domains: a central sequence-specific domain and a C-terminal sequence-independent domain. Here, we show that binding of large but not small DNAs by the C terminus of p53 negatively regulates sequence-specific DNA binding by the central domain. Four previously described mechanisms for activation of specific DNA binding operate by blocking negative regulation. Deletion of the C terminus of p53 activates specific DNA binding only in the presence of large DNA. Three activator molecules (a small nucleic acid, a monoclonal antibody against the p53 C terminus, and a C-terminal peptide of p53) stimulate sequence-specific DNA binding only in the presence of both large DNA and p53 with an intact C terminus. Our findings argue that interactions of the C terminus of p53 with genomic DNA in vivo would prevent p53 binding to specific promoters and that cellular mechanisms to block C-terminal DNA binding would be required.

Replication but Not Transcription of Simian Virus 40 DNA Is Dependent on Nuclear Domain 10

ABSTRACT DNA viruses from several families including herpes simplex virus type 1, adenovirus type 5, and simian virus 40 (SV40), start their transcription and replication adjacent to a specific nuclear domain, ND10. We asked whether a specific viral DNA sequence determines the location of these synthetic activities at such restricted nuclear sites. Partial and overlapping SV40 sequences were introduced into a β-galactosidase expression vector, and the β-galactosidase transcripts were localized by in situ hybridization. Transcripts derived from control plasmids were found throughout the nucleus and at highly concentrated sites but not at ND10. SV40 genomic segments supported ND10-associated transcription only when the origin and the coding sequence for the large T antigen were present. When the large T-antigen coding sequence was eliminated but the T antigen was constitutively expressed in COS-7 cells, the viral origin was sufficient to localize transcription and replication to ND10. Deletion analysis showed that only the large T-antigen binding site II (the core origin) was required but the T antigen was needed for detectable transcription at ND10. Large T antigen expressed from plasmids without the viral core origin did not bind or localize to ND10. Blocking of DNA replication prevented the accumulation of transcripts at ND10, indicating that only sites with replicating templates accumulated transcripts. Transcription at ND10 did not enhance total protein synthesis of plasmid transcripts. These findings suggest that viral transcription at ND10 may only be a consequence of viral genomes directed to ND10 for replication. Although plasmid transcription can take place anywhere in the nucleus, T-antigen-directed replication is apparently restricted to ND10.

Synergistic transcriptional activation of the MCK promoter by p53: tetramers link separated DNA response elements by DNA looping

The WAF1, Cyclin G and muscle creatine kinase (MCK) genes, all contain multiple copies of the consensus p53-binding element within their regulatory regions. We examined the role of these elements in transactivation of the muscle creatine kinase (MCK) gene by p53. The MCK promoter possesses distal (−3182 to −3133) and proximal (−177 to −81) p53-binding elements within which residues −3182 to −3151 (distal) and −176 to −149 (proximal) show homology to the consensus p53-binding site. Using promoter deletion studies, we find that both proximal and distal elements are required for high level, synergistic transcriptional activation in vivo. Electron microscopy indicates that p53-p53 interactions link proximal and distal p53-binding elements and cause looping out of intervening DNA, suggesting that this DNA sequence may be dispensable for synergy. This idea was confirmed by progressive deletion of the DNA between p53-binding elements. Synergism persisted with spacing reduced to only 150 bp. Tetramerization-deficient p53 mutants were defective for transcriptional activation but still capable of synergy. Our results provide evidence for a model by which high level transcriptional activation of promoters with multiple p53 response elements is achieved.

Alternative interactions of the SV40 a protein with DNA

Abstract We have characterized the DNA binding properties of SV40 A protein (T antigen) from permissive cells. The viral protein protects 110- to 115-, 60- to 65-, 40- to 45-, and 30- to 35-bp fragments of SV40 DNA from an excess of DNase 1. The 60- to 65-bp fragment extends some 30–35 by to either side of the Bgl I site at the origin of replication. DNase footprinting confirms the location of this binding region and shows that it is the most completely protected DNA binding site under our conditions. The A protein selectively recognizes the 60- to 65-bp origin fragment in the complete absence of adjacent DNA and binds to it in three alternative ways that protect 60–65, 40–45, or 30–35 by from nuclease. Thus these three interactions with isolated origins recapitulate the binding of viral protein to intact SV40 DNA. We propose that the alternative interactions of SV40 A protein with the 60- to 65-bp origin fragment may represent three different conformational alignments of protein with DNA rather than a simple repetition of a single kind of binding interaction. Viral protein also protects pBR322 and λ phage DNA from nuclease to a limited extent. Similar interactions could possibly alter the function of DNA control regions of host cells.

Binding of dephosphorylated a protein to SV40 DNA

Abstract We have examined the role of SV40 A protein (T antigen) phosphorylation in the specific binding of the protein to SV40 DNA using protein that was dephosphorylated in vitro. Alkaline phosphatase removed more than 75% of the phosphate radiolabel associated with the A protein. Most of the phosphoserine residues were sensitive to the enzyme while most of the phosphothreonine residues were resistant. Phosphopeptide mapping of the dephosphorylated protein demonstrated that the phosphate groups at individual sites were either highly sensitive or highly resistant to the phosphatase. Thus, enzymatically dephosphorylated A protein provides a useful probe for analysis of the function of specific phosphorylation sites. Dephosphorylated A protein, like untreated A protein, preferentially bound a restriction fragment of SV40 DNA that contains the origin of DNA replication and sequences involved in transcriptional control. The binding was characterized further using a DNase protection assay and DNase footprinting. Dephosphorylated A protein protected SV40 DNA in a manner that was indistinguishable from that of untreated protein. The effects of A protein concentration and salt treatment on binding were the same for phosphatase-treated and control untreated A protein under the conditions of our assay. These results suggest that the location, alignment, and affinity of binding are unaffected by dephosphorylation. We conclude that phosphorylation at the majority of the phosphoserine sites is not directly involved in the regulation of site-specific recognition and binding of the A protein to SV40 DNA.

Partial purification of sv40 a protein and a related cellular protein from permissive cells

Abstract A simple and reproducible method for the purification of SV40 A protein (T antigen) from permissive cells has been developed. Two cellular proteins are also purified in smaller quantities by the same procedure. The cellular proteins have molecular weights similar to undegraded SV40 A protein. Peptide maps of the viral and cellular proteins are not similar but one of the cellular proteins apparently shares limited antigenicity with the SV40 A protein.

Identification of the human papovavirus T antigen and comparison with the simian virus 40 protein A

Abstract The simian virus 40 (SV40) A protein (T antigen) and the early proteins produced by cells infected with BKV-type human papovaviruses have been compared by immunoprecipitation and tryptic peptide analyses. Cells infected with the human viruses and transformed cell lines synthesize phosphorylated proteins that interact with antiserum against the SV40 T antigen. These proteins cannot be distinguished from the SV40 A protein by sodium dodecyl sulfate-gel electrophoresis. Peptide map analyses have shown that the human virus proteins are similar, but not identical, to the SV40 A protein. Because of the distinct, reproducible differences in the peptides of the papovavirus proteins, structural analyses provide a direct procedure for detecting the expression of each type of virus in cells.

The role of operator position in SV40 T-antigen-mediated repression

We have manipulated positional relationships between the SV40 early operator and promoter to study the repression of transcription by T antigen. Single or multiple insertions of T-antigen-binding region I resulted in only weak repression even when the operator was placed immediately adjacent to known promoter elements. The low levels of repression appear to reflect the intrinsic strength of the T-antigen-operator interaction.