Bernard Révet

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non‐denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a ‘tandem’ structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a ‘sandwich’ structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.

Four dimers of λ repressor bound to two suitably spaced pairs of λ operators form octamers and DNA loops over large distances

Abstract Transcription factors that are bound specifically to DNA often interact with each other over thousands of base pairs [1,2]. Large DNA loops resulting from such interactions have been observed in Escherichia coli with the transcription factors deoR [3] and NtrC [4], but such interactions are not, as yet, well understood. We propose that unique protein complexes, that are not present in solution, may form specifically on DNA. Their uniqueness would make it possible for them to interact tightly and specifically with each other. We used the repressor and operators of coliphage λ to construct a model system in which to test our proposition. λ repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration. We predict that two λ repressor dimers form a tetramer in vitro when bound to two λ operators spaced 24 bp apart and that two such tetramers interact to form an octamer. We examined, in vitro , relaxed circular plasmid DNA in which such operator pairs were separated by 2,850 bp and 2,470 bp. Of these molecules, 29% formed loops as seen by electron microscopy (EM). The loop increased the tightness of binding of λ repressor to λ operator. Consequently, repression of the λ PR promoter in vivo was increased fourfold by the presence of a second pair of λ operators, separated by a distance of 3,600 bp.

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

The Bacillus subtilis LrpC protein belongs to the Lrp/AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structure-specific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosome-like structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy.

Chromatin reconstitution on small DNA rings. I

Chromatin was reconstituted using the four core histones on 359 base-pair nicked and closed rings by salt dialysis and/or at physiological ionic strength by means of polyglutamic acid. The products, which consisted of mono- and dinucleosomes, were characterized by gel electrophoresis, sedimentation in sucrose gradients and high-resolution electron microscopy. The results were as follows. (1) The efficiency of the reconstitution was found first to increase with the negative linking difference of the closed rings relative to their relaxed configuration to reach a maximum for -2 turns, and then to decrease for the largest difference of -3 turns. Discrepancies between topoisomers were also observed with regard to differential formation of mono- and dinucleosomes. Topoisomer -1 reconstituted monomers easily but reconstituted dimers with difficulty, whilst this discrimination was virtually absent in the case of topoisomers -2 and -3. Moreover, mononucleosomes on the nicked ring were, with respect to their electrophoretic mobility, similar to mononucleosomes formed on topoisomer -1 but not to those on the other topoisomers, whose mobilities were greater. These features were interpreted in terms of the linking number change associated with the formation of a nucleosome monomer and dimer, approximately -1 and -2 turns, respectively. (2) Two dinucleosome subtypes were found to form in a sequential manner. Their different electrophoretic mobilities and sedimentation coefficients suggested that the early subtype is lighter, probably because of an incomplete histone complement in the second nucleosome of that subtype as a result of an impaired co-operativity in octamer assembly due to the small ring size. (3) An electron microscopic examination of the chromatin reconstituted on topoisomer -2 revealed that both mono- and dinucleosomes adopt two different, salt-dependent, morphologies each: in type I, entering and exiting DNAs do not cross, whilst they do in type II. Type I configuration is favoured in lower salt, whereas type II is favoured in higher salt. Such behaviour explains why nucleosomes in dimers were found to be always diametrically opposed on the rings rather than sometimes apposed, as would have been expected from a random deposition of the histone cores.

The Drosophila X virus contains a 1-μM double-stranded RNA circularized by a 67-kd terminal protein: High-resolution denaturation mapping of its genome

Abstract The effect of urea on Drosophila X virus (DXV) is presented and analyzed by electron microscopy. At 4 M urea a RNA-protein complex is liberated consisting of one segment of double-stranded (ds) RNA which is maintained in a circular form inside the virion by a protein of 67 kd. The RNA-protein complex was purified by chromatography and had a tendency to form very spectacular flower-like structures. By crosslinking the RNA inside the virus with psoralen and uv radiation it is shown that the RNA was double-stranded in situ. A new method for partial denaturation mapping of double-stranded nucleic acids by electron microscopy is described and was applied to DXV RNA. Under these conditions it is shown that native and denatured regions as small as 30 RNA base pairs (bp) can be visualized. In the case of DXV RNA it was observed that the RNA possessed at one extremity a GC-rich region of 30 by followed by an AU-rich region of 160 bp. These molecules were observed in a conventional transmission electron microscope and a scanning transmission electron microscope under dark-field illumination. Absolute electron microscope length measurements and molecular weight determinations by gel migration revealed that the DXV RNA is one segment of ds-RNA of 0.97 μm in the electron microscopic conditions used in this work, giving a molecular weight of 2.2 × 106 d which corresponds to 3170 RNA bp. An average melting temperature of 83.3° was obtained in 0.1 SSC. Viral protein analysis was performed on the virus and on the purified protein-RNA complex. The virus is made of six major proteins and two minor proteins which were revealed after in vitro iodination.

Circular adenovirus DNA-protein complexes from infected HeLa cell nuclei

Adenovirus (Ad) DNA was extracted by the use of Sarkosyl from Ad5-infected HeLa cell nuclei at 15 hr after infection. Mature and replicating viral DNA molecules in the Sarkosyl extract were purified by sucrose gradient centrifugation followed by equilibrium centrifugation in preformed metrizamide gradients. Part of the material was digested with the Hpa I restriction endonuclease and analyzed by electrophoresis through 1% agarose gels. Both terminal fragments D and E did not enter the gel unless first treated with Pronase, suggesting that intracellular viral DNA molecules were linked to proteins by their termini. Examination under the electron microscope of mature intracellular Ad2 or Ad5 DNA molecules extracted with Sarkosyl showed the presence of a majority of circular unit-length molecules. Most of these were attached to a prominent globular structure which disappeared, together with the circularization of the molecule, after treatment with Pronase. We interpret these structures as protein links responsible for the circularization of the molecule. The mature linear DNA molecules observed also showed evidence for linkage structures attached to one or both of their ends. Examination of replicating Ad5 DNA molecules extracted with Sarkosyl revealed the presence of various types of forked, circular molecules, most of which showed evidence for the presence of linkage structures. It is suggested that replicating and newly made Ad DNA molecules are circular inside the cell, perhaps through linkage with proteins, and the possible role of circularization in the replication of viral DNA is discussed.

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

Tn4430 is a distinctive transposon of the Tn3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro. Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/or DR2, TnpI‐mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild‐type IRSs exclusively produces two‐noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented.

Characterization of a new adenovirus type 5 assembly intermediate.

Adenovirus H5 (Ad5) DNA-protein complexes were extracted with ammonium sulphate (0.2 M) from virus-infected HeLa cell nuclei at 18 h after infection. Analysis of the material by centrifugation through discontinuous sucrose gradients in heavy water revealed the existence of several populations of molecules which were identified, in order of increasing buoyant density, as mature DNA-protein complexes, replication complexes, assembly intermediates and virions. When observed under the electron microscope, some of the assembly intermediates showed a capsid with a tail of entirely double-stranded (ds) DNA, or of dsDNA continued by a portion of single-stranded (ss) DNA thickened by a coat of E-72 K DNA binding protein. Singly or doubly-forked Ad5 replicating DNA molecules partially packaged in virus capsids were also observed. It is suggested that these molecules could be assembly intermediates, i.e. one of the first steps of assembly corresponding to virus DNA entering pre-formed capsids or their precursors. The fact that replication was still going on at one end of many of the DNA molecules in the intermediates, while encapsidation was taking place at the other, raises the possibility of a coupled DNA replication-packaging process in the formation of adenovirions.

Electron microscopy of AD5 replicating molecules after in vivo photocrosslinking with trioxsalen

Abstract A new approach for studying replicating DNA structures is presented. Ad5 replicative intermediates were fixed in vivo by inducing DNA interstrand crosslinks with 4,5′,8-trimethylpsoralen and near ultraviolet light (365 nm) in order to maintain the structure of replicating DNA molecules and to prevent branch migration which may occur during the isolation and analysis of these molecules. The replicating Ad5 material was analyzed under neutral and denaturing conditions by electron microscopy. The in vivo crosslinking followed by a mild extraction of nuclei allowed intact replicating molecules to be isolated. In this way all branch positions characteristic for the first round of replication were observed. Furthermore, a statistical analysis of more than a hundred molecules with multiple single-stranded branches visualized by electron microscopy showed that growing points move from one end of the Ad5 molecule toward the other one and not in opposite directions. Crosslinking of the duplex part of the replicating molecules made it possible to visualize these structures for the first time under denaturing conditions. The direction of DNA synthesis at the growing points can be specified and replication is shown to occur continuously.

Three Dimensional Association of Double-Stranded Helices Are Produced in Conditions for Z-DNA Formation

The Z form of alternating poly(dG-dC).poly(dG-dC) can be induced when the concentration of NaCl, MgCl2 or ethanol are increased. In order to obtain more information concerning this Z structure, the B----Z transition is analyzed on the same sample, both by UV spectrophotometry and electron microscopy. The procedures used in this work provide high resolution images with minimal alterations of the molecules. It is shown that at high values of cations or ethanol, the polymer makes complex associations of numerous molecules stuck together parallelly. By decreasing the salt or ethanol concentrations, a progressive decondensation of the molecules is obtained. At low concentrations of Mg++ (2.10(-2) M), alterations of the linear secondary structure of the molecules are observed, although the UV spectrum is of the B-type. In the presence of that low concentration of Mg++, natural DNAs (phi X174 and yeast mitochondrial DNA fragment inserted in pBR) exhibit structural modifications similar to those observed with the poly(dG-dC).poly(dG-dC). These structures mainly consist in four-stranded hairpins and loops built up by the sticking of two segments of DNA. The correlation between these intertwining of short DNA segments and the presence of potentially Z-forming sequences is discussed.