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P V Hough

Publications and source records attributed to P V Hough.

13 recordsLinked to original sources
PubMed1994

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.

Animals↗
PubMed1994

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.

Antigens, Viral, Tumor↗
PubMed1993

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.

Animals↗
PubMed1991

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.

DNA↗
PubMed1989

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.

Adenosine Triphosphate↗
PubMed1987

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.

Adenoviridae↗
PubMed1985

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.

Antigens, Polyomavirus Transforming↗
PubMed1981

Excitation under the scanning electron microscope of DNA-associated fluorescence from chicken erythrocyte nuclei, polytene chromosomes and adenovirus 2 virions.

Biological structures not seen by conventional light microscopy, such as longitudinal striations in polytene chromosomes, and, at the limit of sensitivity, virions of adenovirus 2, have been detected via DNA-associated fluorescence excited under the scanning electron microscope. The maximum sensitivity realized, about 1 detected photon per 700 base pairs, falls short by about an order of magnitude of that required to achieve, in unreplicated specimens, the 2 nm intrinsic resolution of the method. A combination of D2O-H2O substitution with freeze-drying provides the best unquenching procedure found for in situ DNA. DNA-associated fluorescence for light microscopy can be created by moderate exposure of the specimen in the electron microscope.

Adenoviruses, Human↗
PubMed1976

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifidable in situ via the fluorescence excited by the focused electron beam of a canning electron microscope. A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam. Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules. Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues. The theoretical resolution limit for localization of a particular molecular species -- about 20 A--is determined by the known maximum distance for molecular excitation by fast electrons. Drect extapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A. Fluorescence is strongly quenched by residual water in the specimen. Similar quenching is produced by some macromolecular associations and so may serve to localize such associations.

Animals↗
PubMedDate not supplied

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.

Bacteriophage T4↗