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.

The HMG-box mitochondrial transcription factor xl-mtTFA binds DNA as a tetramer to activate bidirectional transcription.

The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided evidence of cross-linked dimers, trimers and tetramers of xl-mtTFA. Finally, high resolution scanning transmission electron microscopy (STEM) established that xl-mtTFA binds to the specific promoter-proximal site predominantly as a tetramer. Computer analysis of several previously characterized binding sites for xl-mtTFA revealed a fine structure consisting of two half-sites in a symmetrical orientation. The predominant sequence of this dyad symmetry motif shows homology to binding sites of sequence-specific HMG-box-containing proteins such as Sry and Lef-1. We suggest that bidirectional activation of transcription results from the fact that binding of a tetramer of xl-mtTFA permits symmetrical interactions with other components of the transcription machinery at the adjacent core promoters.

Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase.

Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.

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.

Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy.

For inorganic crystals such as calcite (CaCO3), Atomic Force Microscopy (AFM) has provided surface structure at atomic resolution (Ohnesorge and Binnig, 1993). As part of a broad effort to obtain high resolution for an individual protein or protein assembly (Binnig et al., 1986; Rugar and Hansma, 1990; Radmacher et al., 1992), we applied AFM to study the ATP-dependent double hexamer of SV40 large T antigen, which assembles around the viral origin of DNA replication. Multimeric mass has been determined in two-dimensional projected images by Scanning Transmission Electron Microscopy (STEM) (Mastrangelo et al., 1989). By AFM, if the DNA-protein preparation has been stained positively by uranyl acetate, the contour at the junction between hexamers is visible as a cleft, 2-4 nm deep. The cleft, whether determined as a fraction of height by AFM or as a fraction of mass thickness by STEM, is of comparable magnitude. On either side of the cleft, hexamers attain a maximum height of 13-16 nm. Monomers found in the absence of ATP show heights of 5-7 nm. Taken together, the z coordinates provide a surface profile of complete and partial replication assemblies consistent with the spatial distribution of recognition pentanucleotides on the DNA, and they contribute direct geometrical evidence for a ring-like hexamer structure.

Recent advances in atomic force microscopy of DNA.

Three advances involving DNA in atomic force microscopy (AFM) are reported here. First a HEPES-Mg buffer has been used that improves the spreading of DNA and provides good DNA coverage with as little as 200-500 picograms per sample. Second, the new "tapping" mode has been used to improve the ease and resolution of AFM-imaging of DNA in air. Finally, AFM images are presented of single-stranded phi X-174 virion DNA with the gene 32 single-stranded binding protein. A summary of the current state of the field and of the methods for preparing and imaging DNA in the AFM is also presented.

RIP60 dimers and multiples of dimers assemble link structures at an origin of bidirectional replication in the dihydrofolate reductase amplicon of Chinese hamster ovary cells.

We show assembly of low and high multimers of HeLa cell nuclear protein, RIP60, at the origin of bidirectional replication (OBR) identified by Burhans, Vassilev, Caddle, Heintz and DePamphilis in Chinese hamster ovary cells. RIP60 binds a 5'-ATT-3' reiterated sequence downstream of the OBR and a second, homologous ATT sequence of opposite orientation situated within the OBR zone. Specifically bound structures were studied by conventional electron microscopy (EM) and quantitative scanning transmission electron microscopy (STEM). Dimers and multiples of dimers link the downstream binding site that overlaps a bent DNA sequence and the homologous upstream OBR sequence, looping out 700 bp of intervening DNA. Superposed dimers are found at individual unlinked sites, stabilized presumably through protein-protein interaction, and such superposition appears to occur also in the basic link structure. Along the loop, single crossovers and extended twists are observed by conventional EM. By STEM, loop DNA is laterally compacted, with diameter and mass equivalent to double-duplex DNA strands. Supercoiled 736 bp and 5243 bp circular DNAs assume similar laterally compacted geometries that are mostly absent from relaxed forms. These observations parallel the compacted, interwound superhelices viewed by cryo-electron microscopy in vitrified solutions containing magnesium ions, and provide structural evidence in agreement with that from conventional EM for superhelical tension in RIP60 loop DNA. Loop superhelicity could arise as a topological response to linking and suggests a functional role for link formation.

DNA looping and Sp1 multimer links: a mechanism for transcriptional synergism and enhancement.

Using conventional and scanning transmission electron microscopy, we have examined the physical basis of long-range enhancer effects between distal and proximal elements in a eukaryotic promoter. Specifically, we have studied binding of human transcription factor Sp1 to 10-base-pair G+C-rich elements ("GC boxes") located at -100 and +1700 relative to the RNA start site. It was previously observed that the distantly located site functions in synergism with the promoter-proximal site to strongly activate transcription in vivo. Here we demonstrate that this synergism is likely to be a direct consequence of interactions between remote and local Sp1, the remote Sp1 translocated to the promoter by a DNA loop. Scanning transmission electron microscopy shows that Sp1 initially forms a tetramer and subsequently assembles multiple tetramers stacked in register at the DNA loop juncture. This unexpected finding not only provides the physical basis for loop formation but also defines a biological process leading to strongly increased concentration of activator protein at the promoter. The mechanism may unify the problem of transcriptional activation by removing enhancer action as a separate class of regulatory activity.

ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication.

Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.

The gene-specific initiation factor USF (upstream stimulatory factor) bound at the adenovirus type 2 major late promoter: mass and three-dimensional structure.

The gene-specific transcription initiation factor USF (upstream stimulatory factor) binds at a palindromic sequence that extends from -52 to -63 relative to the start site of the adenovirus type 2 major late promoter; USF enhances in vitro transcription 10- to 20-fold. By analysis of digital micrographs from the Brookhaven scanning transmission electron microscope, we have identified a sample of 29 proteins (mass, 55 +/- 5 kDa) specifically bound at the palindrome. The individual protein digital images show extensive homology, which permits modeling a three-dimensional structure at a relatively low resolution, which is nonetheless significant for the study of protein-protein interactions in initiation. Non-sequence-specific competitor DNA at high mass excess can be used in reactions for microscopy, enabling characterization of specific binding for proteins present at 1% of total protein or less.

Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen.

Three sequence components direct high affinity binding of dimeric SV40 T antigen to SV40 origin region I. Two signals are encoded by two directly repeated 5'-GAGGC-3' pentanucleotides. Approximately equal contributions to binding stability are made by each pentanucleotide, and both spacing and orientation of the pentanucleotides are important for binding affinity. The third vital component is contained in a 5'-TTTTTTG-3' spacer sequence that separates the pentanucleotides. Sequence-specific features of the spacer stabilize binding to the adjacent pentanucleotides. The asymmetry of the spacer suggests that a novel binding mechanism is involved. Because the alignment of T antigen on mutant and wild-type DNAs is similar, we propose that any two of the three sequence signals are sufficient to determine the unique arrangement of a bound protein dimer.

Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution.

Large tumor (T) antigen and its bound multimeric states are positioned by scanning transmission electron microscopy (STEM) within a few base pairs at control sequences of the simian virus 40 DNA origin of replication region. Proximal and distal edge positions for each multimer group match the end positions of previously mapped fragments protected from DNase cleavage. Since chance correspondence is shown to be extremely unlikely, STEM mass measurements, obtained concurrently with STEM map positions, indicate that the DNase fragments arise from bound monomers, dimers, trimers, and tetramers in binding region II and monomers, dimers, and trimers in binding region I. Simultaneous binding of seven monomer-equivalent masses is observed, three in region I and four in region II, with an ordered and interpretable mass distribution in the plane of the foil. Although this observation does not prove that the six G-A-G-G-C and one T-A-G-G-C sequences, similarly distributed, function as recognition sequences for T-antigen monomer, it provides strong support for such a model. The stable existence in solution of low-and intermediate-mass structures, observed at lower T-antigen concentrations, suggests a role as assembly intermediates.

Chinese hamster ovary chromosomes and antigens in tobacco/hamster heterokaryons.

Tobacco GGLL/hamster CHO hamster heterokaryon frequency following cell fusion has been raised to 4-10 percent with a modified PEG procedure. In heterokaryons, GGLL nuclei become activated, adhere to and appear to fuse with CHO nucleo. Metaphase CHO chromosomes can be identified 24 hours after fusion and immunofluorescent CHO cell surface antigens diffuse into the plant cell membrane. Diffusion coefficients, D, are estimated to be between 0.6 X 10(-11) and 1.6 X 10(-11) cm2sec-1.

Interkingdom fusion between human (HeLa) cells and tobacco hybrid (GGLL) protoplasts.

The fusion of human HeLa cells with tobacco protoplasts has been accomplished with the use of polyethylene glycol. The sequence from heterocellular adherence to heterokaryon formation has been followed with light microscopy and confirmed by autoradiographs of heterokaryons containing unlabeled tobacco nuclei and tritium-labeled HeLa nuclei. The HeLa nucleus retained its integrity in the tobacco cytoplasm up to 6 days after fusion.