Nucleosome dynamics. Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)2 tetramer-DNA particle.

Our laboratory has previously reported the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)2tetramer). This transition was induced by DNA positive torsional constraint, generated either by initial supercoiling of the loop or by its thermal fluctuations during topoisomerase relaxation. Taking into account the wrapping of the DNA around the histones into less than a turn, and its negative crossing at the entry-exit, the transition was proposed to involve a 360 degrees rotation of the loop around the particle dyad axis, and the formation of a positive crossing. The tetramer horseshoe-shaped conformation within the octamer further suggested that this process could be mediated by a reorientation of the two sector-like H3-H4 dimers about their H3/H3 interface, which would switch the overall handedness of the proteinaceous superhelix from left to right-handed. We now provide additional evidence for such a contribution of the protein by showing, through gel electrophoresis, topoisomerase relaxation and electron microscopy, that a sterical hindrance at the H3/H3 interface, introduced by covalent linking of bulky adducts through thiol oxidation of H3 cysteine 110, interferes with the transition. Such interference varies, depending on the particular SH-reagent used; but the most remarkable effect was obtained with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB), which displaces the preferred conformation of the tetrasomes from left-handed to semi-right-handed, and at the same time preserves a significant degree of chiral flexibility. DNA contribution was evidenced by a specific fractionation of circular tetrasomes in gel electrophoresis which, together with a different positioning of control and DTNB tetrasomes on linear DNA, pointed to an interdependence between tetrasome conformation and positions. Moreover, linear tetrasomes fluctuate between crossed and uncrossed conformations in a salt-dependent equilibrium which appears to vary with their positions on the DNA. These data suggest a modulatable role of the DNA around the dyad in the transition, depending primarily on its sequence-dependent deformability. This role is played at both levels of H3-H4 dimer reorientation and lateral opening, a mechanism by which the particle may relieve the clash between its entering and exiting DNAs. These properties make the tetrasome an attractive potential intermediate in nucleosome dynamics in vivo, in particular duringX transcriptional activation and elongation.

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

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 lambda to construct a model system in which to test our proposition. lambda repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration. We predict that two lambda repressor dimers form a tetramer in vitro when bound to two lambda 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 lambda repressor to lambda operator. Consequently, repression of the lambda PR promoter in vivo was increased fourfold by the presence of a second pair of lambda operators, separated by a distance of 3,600 bp.

Isolation and characterization of a protein with high affinity for DNA: the glutamine synthetase of Thermus thermophilus 111.

In a search of proteins from the thermophilic bacterium Thermus thermophilus 111 with a high affinity for DNA, the selected protein from this screening appears to be the glutamine synthetase (GS). The purified product gives one band in SDS-polyacrylamide gel electrophoresis (53,700 Da). The N-terminal 32 residues have been identified and present an homology of 80% with the glutamine synthetase of Bacillus subtilis and 76% with that of Thermotoga maritima. The protein displays the characteristic dodecameric structure of the eubacteria glutamine synthetase. From a detailed study of the interaction of this protein with DNA by dark-field electron microscopy and agarose gel electrophoresis, it is concluded that double-stranded DNA wraps the protein by a full turn of 150 bp length. An even number of GS molecules bound to a closed relaxed plasmid DNA does not alter its null topology. By using an inverted dimer DNA fragment, which contains twice a curved kinetoplast DNA insert in its central part, it is shown that DNA curvature rules the order in which GS binds to the DNA. DNA ends are also sites of high affinity for the GS. Supercoiling does not favor the binding of GS to the DNA with the exception of the apices that are by essence bent regions. By saturating a DNA molecule with GS one obtains a novel characteristic scalloped configuration in which the DNA undulates from one GS to the next. The DNA is condensed at least three times in these structures. By increasing the ratio of GS to DNA in solution the resulting material migrates as discrete bands relative to the free DNA in an agarose gel. By gel retardation and EM statistical distribution analysis of GS within the complexes, an average affinity constant of 10(7) M-1 was obtained. The potential implications of this novel interaction of the glutamine synthetase with DNA for the regulation of its own gene are briefly discussed.

Nucleosome dynamics. II. High flexibility of nucleosome entering and exiting DNAs to positive crossing. An ethidium bromide fluorescence study of mononucleosomes on DNA minicircles.

H2A-H2B exchange with the intranuclear histone pool upon chromatin transcription in vivo is generally viewed as being triggered by the DNA positive supercoiling wave pushed by the elongating polymerase. This notion was tested here by investigating a potential release of H2A-H2B by ethidium bromide-induced positive supercoiling in the loop of mononucleosomes reconstituted on DNA minicircles. The results of gel electrophoresis, fluorescence titration and electron microscopy showed that such a positive supercoiling was not able to release H2A-H2B, nor to unfold the nucleosome to any detectable extent. The reason appeared to be the ease with which the loop could undergo a positive crossing, a surprising observation in view of the DNA left-handed wrapping around the octamer. Moreover, the influence of histone acetylation suggested that such loop flexibility to positive crossing is mediated by histone N-terminal tails which, by interacting with entering and exiting DNAs, reduce their electrostatic repulsion. These conclusions are confirmed and extended in the accompanying article through relaxation with topoisomerase I.

Short unligated sticky ends enable the observation of circularised DNA by atomic force and electron microscopies.

A comparative study of the stabilisation of DNA sticky ends by divalent cations was carried out by atomic force microscopy (AFM), electron microscopy and agarose gel electrophoresis. At room temperature, molecules bearing such extremities are immediately oligomerised or circularised by addition of Mg2+or Ca2+. This phenomenon, more clearly detected by AFM, requires the presence of uranyl salt, which stabilises the structures induced by Mg2+or Ca2+. DNA fragments were obtained by restriction enzymes producing sticky ends of 2 or 4 nucleotides (nt) in length with different guanine plus cytosine (GC) contents. The stability of the pairing is high when ends of 4 nt display a 100% GC-content. In that case, 95% of DNA fragments are maintained circular by the divalent cations, although 2 nt GC-sticky ends are sufficient for a stable pairing. DNA fragments with one blunt end and the other sticky appear as dimers in the presence of Mg2+. Dimerisation was analysed by varying the lengths and concentrations of DNA fragments, the base composition of the sticky ends, and also the temperature. Our observation provides a new powerful tool for construction of inverted dimers, and circularisation, ligation analysis or short bases sequence interaction studies.

Interaction of the histone (H3-H4)2 tetramer of the nucleosome with positively supercoiled DNA minicircles: Potential flipping of the protein from a left- to a right-handed superhelical form.

We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two H3s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a left- to a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E. & Moudrianakis, E. N. (1991) Proc. Natl.Acad. Sci. USA 88, 10148-10152] that this change in DNA topology is mediated by a similar change in the topology of the tetramer itself, which may occur through a rotation (or a localized deformation) of the two H3-H4 dimers about their H3-H3 interface. Potential implications of this model for nucleosome dynamics in vivo are discussed.

Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation.

Electron microscopic visualization indicates that the transcription activator NRI (NTRC) binds with exceptional selectivity and efficiency to a sequence-induced superhelical (spiral) segment inserted upstream of the glnA promoter, accounting for its observed ability to substitute for the natural glnA enhancer. The cooperative binding of NRI to the spiral insert leads to protein oligomerization which, at higher concentration, promotes selective coating of the entire superhelical segment with protein. Localization of NRI at apical loops is observed with negatively supercoiled plasmid DNA. With a linear plasmid, bending of DNA is observed. We confirm that NRI is a DNA-bending protein, consistent with its high affinity for spiral DNA. These results prove that spiral DNA without any homology to the NRI-binding sequence site can substitute for the glnA enhancer by promoting cooperative activator binding to DNA and facilitating protein oligomerization. Similar mechanisms might apply to other prokaryotic and eukaryotic activator proteins that share the ability to bend DNA and act efficiently as multimers.

Taxonomic studies of Rickettsiella, Rickettsia, and Chlamydia using genomic DNA.

Invertebrate pathogens of the genus Rickettsiella (Order Rickettsiales) undergo a developmental cycle more characteristic of chlamydial organisms (Order Chlamydiales) than of typical rickettsiae. Moreover, among recognized species of Rickettsiella, there is considerable variation in host range and in the ultrastructure and development of the infectious stage, i.e., the elementary body. To begin an analysis of the taxonomic relationships of the invertebrate pathogens belonging to the genus Rickettsiella and of the relationship of these to chlamydiae and other rickettsiae, genomic DNA of representative species was compared with respect to restriction enzyme site polymorphism, DNA-DNA hybridization, G + C ratios, and fine melting profiles. The strains studied included isolates currently classified as Rickettsiella grylli, R. popilliae, R. chironomi, Chlamydia psittaci, C. trachomatis, Coxiella burnetii, and Rickettsia conorii. Reciprocal DNA hybridization studies carried out under high-stringency conditions (65 degrees C) showed homology between R. grylli and R. popilliae isolates but not with the others, including two isolates of R. chironomi from the midge, Chironomus dorsalis, and the scorpion, Buthus occitanus. The R. chironomi isolates shared no detectable homology with each other or with any of the other strains or species. Additionally, no homology was detected between any of the Rickettsiella isolates and the species of Coxiella, Rickettsia, or Chlamydia. Results obtained from the DNA fine melting profiles and G + C ratios corresponded with the results obtained from the DNA hybridization studies. Thus, the present study indicates that, despite similarities in developmental cycles, Rickettsiella and Chlamydia are taxonomically distinct groups appropriately placed in different orders.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational analysis of a 139 base-pair DNA fragment containing a single-stranded break and its interaction with human poly(ADP-ribose) polymerase.

The conformational changes induced by the introduction of a central and unique single-stranded break in a 139 base-pair DNA duplex have been analysed by means of polyacrylamide gel electrophoresis, HPLC and dark-field electron microscopy. Compared to the control DNA, the disruption of the covalent sugar-phosphate backbone induces a retardation detected both by gel electrophoresis and anion exchange based HPLC. Electron microscopic visualization of the DNA molecules reveals that most of them present a central fracture at the position of the nick. Measures of the angle at the apex were very well fitted by a simple model of isotropic flexible junction assuming spatial Hooke's law and simple basic Boltzmann statistics. This amounts to using a folded Gaussian distribution. The fit yields an angle equilibrium value phi 0 = 122 degrees for the nicked fragment. The angle distribution could also result from an equilibrium between two forms of the molecule with isotropic flexibility at the nicked site: a stacked and a very flexible unstacked form. The majority of bound poly(ADP-ribose) polymerase, a zinc-finger enzyme involved in DNA break detection, was localized at the apex of the V-shaped DNA duplex, with an accentuation of its general V-shaped conformation (phi 0 = 102 degrees).

Homologous DNA targeting with RecA protein-coated short DNA probes and electron microscope mapping on linear duplex molecules.

We demonstrate that RecA protein-coated, short single-stranded DNA probes paired with a specific homologous DNA sequence in a linear duplex target molecule and accurately targeted the selected DNA sequence. RecA protein-coated complementary ssDNA probes were reacted with linear duplexes, and the homologously paired molecules were observed by electron microscopy. The sites of interaction between the RecA protein-coated DNA probes and the uncoated duplex DNA targets were directly visible on individual target DNA molecules by high-resolution darkfield electron microscopy, without chemical fixation or sample shadowing. The efficiency and specificity of pairing were verified with 446 and 222 base single-stranded DNA probes that shared no homology with one another, and several linear duplex target DNAs with their respective probe homology sites at different locations with respect to the ends of the double-stranded DNA molecules. Measurements of the position of RecA protein-coated probes paired to individual target molecules, observed at high magnification, showed that DNA probes specifically paired at their corresponding homologous target sequences. This RecA protein-mediated DNA mapping method allows homologous sequence positioning and gene mapping on individual double-stranded DNA molecules. Targeting reactions in which two different probe/target sites were 900 bases apart on a single duplex target molecule allowed both sites to be mapped in the same targeting reaction; although targets displaying both probes simultaneously were seen much less frequently than expected. The possible torsional or mechanistic constraints related to these reactions are briefly discussed.

Quantitative electron microscopic analysis of DNA-protein interactions.

Electron microscopy offers a unique potentiality to visualize individual molecules. For the last 30 years it has been used to study the structure and the interactions of various biological macromolecules. The contribution of electron microscopy is important because of its capacity to demonstrate the existence of conformational structures such as kinks, bents, loops, etc., either on naked DNA, or on DNA associated with various proteins or ligands. Increasing interest was given to such observations when it was found that they provide a direct visualization of interacting molecules involved in DNA metabolism and gene regulation. Technical advances in the preparation of the specimens, their observation in the electron microscope, and the image processing by computers have allowed the shifting from qualitative to quantitative analysis, as illustrated by a few examples from our laboratory.

Removal of DNA curving by DNA ligands: gel electrophoresis study.

The removal of inherent curving in Crithidia fasciculata kinetoplast DNA by various small DNA ligands, groove binders and mono- and bisintercalators, has been studied by gel retardation and electron microscopy. The migration of the kinetoplast DNA fragment is highly retarded during gel electrophoresis. We demonstrate that this retardation is suppressed by DNA ligands such as distamycin and ditercalinium, which have different modes of binding and sequence specificities. Observation by electron microscopy confirms that the effect of ditercalinium on gel migration of curved DNA is linked to DNA uncurving. As the drug is progressively added to DNA, a large broadening of the retarded band is observed during gel electrophoresis for distamycin and ditercalinium. In the case of distamycin, the retarded DNA band splits into two broad bands, whereas the noncurved DNA bands remain homogeneous. This indicates that the drug-DNA exchange is extremely slow in the gel and that a limited number of specific sites on DNA are critical for the removal of bending. GC-specific quinomycin, monointercalators, and bisintercalators act in a manner similar to that of AT-specific distamycin. This indicates that direct drug binding at the dAn tracts is not required for DNA uncurving. We propose that the uncurving of kinetoplast DNA by drugs is caused by a global alteration of DNA structure; subsequent increased flexibility leads to the suppression of rigid bending at the AT tract junctions.

Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate.

The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D. A., D. J. Arndt-Jovin, M. Robert-Nicoud, L. P. McIntosh, R. Tomae, and T. M. Jovin. 1984. J. Mol. Biol. 176:369-415). Divalent cations are also important in the structure of the quasi-permanently condensed chromosomes of dinoflagellate protists (Herzog, M., and M.-O. Soyer. 1983. Eur. J. Cell Biol. 30:33-41) which also have superhelicity in their DNA. The absence of histones in dinoflagellate chromosomes suggest that the search for Z-DNA sequences might be fruitful and could provide one indication of the physiological role of this particular DNA conformation. We report a complete immunofluorescent and immunogold analysis of the nuclei of the dinoflagellate Prorocentrum micans E. using monoclonal and polyclonal anti-B and anti-Z-DNA antibodies. Positive labeling was obtained with immunofluorescence using squash preparations and cryosections, both of which showed the intranuclear presence of the two DNA conformations. In ultrathin sections of aldehyde-prefixed, osmium-fixed, and epoxy-embedded cells, we have localized B-DNA and Z-DNA either with single or double immunolabeling using IgG labeled with 5- and 7-nm gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their arch-shaped configuration, are abundantly labeled with anti-B-DNA antibody. Extrachromosomal anti-B-DNA labeling is also detected on the nucleoplasm that corresponds to DNA loops; we confirm the presence of these loops previously described external to the chromosomes (Soyer, M.-O., and O. K. Haapala. 1974. Chromosoma (Berl.). 47:179-192). B labeling is also visible in the nucleolus organizer region (NOR) and in the fibrillo-granular area (containing transcribing rDNA) of the nucleolus. Z-DNA was localized in limited areas inside the chromosomes, often at the periphery and near the segregation fork of dividing chromosomes. In the nucleolus, Z-DNA is observed only in the NOR area and never in the fibrillo-granular area. For both types of antibody experiments, controls using gold-labeled IgG without primary antibody were negative. A quantitative evaluation of the distribution of the gold-labeled IgG and a parametric test support the validity of these experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron microscopy mapping of pBR322 DNA curvature. Comparison with theoretical models.

A map of local curvature of the pBR322 DNA has been established by electron microscopy analysis of linearized plasmid molecules. To determine their polarity these molecules are one end labelled with an avidin-ferritin-biotin complex and the images are digitized. Local curvature is calculated from two mathematical treatments of the DNA trajectory and expressed in term of a mean dinucleotide wedge angle. Eight regions of curvature are distinguished. The four main regions of curvature have a high content of phased AA runs. The experimental curvature map is compared to theoretical maps of curvature obtained from four available models for DNA curvature.

Molecular analysis by electron microscopy of the removal of psoralen-photoinduced DNA cross-links in normal and Fanconi's anemia fibroblasts.

The induction and fate of psoralen-photoinduced DNA interstrand cross-links in the genome of Fanconi's anemia (FA) fibroblasts of complementation groups A and B, and of normal human fibroblasts, were investigated by quantitative analysis of totally denatured DNA fragments visualized by electron microscopy. 8-Methoxypsoralen (5 x 10(-5) M) interstrand cross-links were induced as a function of the near ultraviolet light dose. With time of postexposure incubation, a fraction of interstrand cross-links disappeared in all cell lines. However, 24 h after treatment, this removal was significantly lower in the two FA group A cell lines examined (34-39%) than in the FA group B and normal cell lines (43-53 and 47-57%, respectively). These data indicate that FA cells are at least able to recognize and incise interstrand cross-links, as normal cells do, although group A cells seem somewhat hampered in this process. This is in accord with data obtained on the same cell lines using another biochemical assay (D. Papadopoulo, D. Averbeck, and E. Moustacchi. Mutat. Res., DNA Repair Rep., 184: 271-280, 1987). Since the fate of cross-links in FA constituted a controversial matter, it is important to stress that two different methodologies applied to genetically well defined cell lines led to the same conclusions.

Structural modifications induced by the mtDBP-C protein in the replication origin of Xenopus laevis mitochondrial DNA.

The structure of the non-coding region of Xenopus laevis mitochondrial DNA has been studied by electron microscopy analysis of DNA molecules end-labelled with streptavidin-ferritin. We have shown that the effect of a protein modifying the shape of the DNA double-helix can be studied and precisely located by this method. It was found that the non-coding region contains curved segments and that the mitochondrial protein mtDBP-C preferentially enhances the curvature of the promoters-replication origin region.

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.

Chromatin reconstitution on small DNA rings. II. DNA supercoiling on the nucleosome.

DNA supercoiling on the nucleosome was investigated by relaxing with topoisomerase I mono- and dinucleosomes reconstituted on small DNA rings. Besides 359 base-pair (bp) rings whose linking differences were integers, two additional series of rings with fractional differences, 341 and 354 bp in size, were used. Mononucleosomes reconstituted on 359 bp rings were found to relax into a single mononucleosome form. In contrast, 341 and 354 bp mononucleosomes relaxed into a mixture of two forms, corresponding to two adjacent topoisomers. The observation that the ratio between these two forms was, within each ring series, virtually independent of the initial linking number of the topoisomer used for the reconstitution suggested that each partition reflected an equilibrium. Comparison with the equilibria observed for the same rings in the absence of histones showed that the formation of a single nucleosome is associated with a linking number change of -1.1(+/-0.1) turn. Dinucleosomes, in contrast, were not relaxed to completion and do not reach equilibria. The corresponding linking number change per nucleosome was, however, estimated to be similar to the above figure, in agreement with previous data from the literature obtained with circular chromatins containing larger numbers of nucleosomes. DNA structure in mononucleosomes was subsequently investigated by means of high-resolution electron microscopy and gel electrophoresis. It was found that the above linking number reduction could be ascribed to a particle with a large open extranucleosomal DNA loop and with no more than 1.5 turns of a superhelix around the histone core. A theoretical model of a nucleosome on a small ring was constructed in which one part of the DNA was wrapped around a cylinder and the other part was free to vary both in torsion and flexion. The linking number reduction predicted was found to be most consistent with experimental data when the twist of the DNA in the superhelix was between 10.5 and 10.65 pb per turn, suggesting that wrapping on the nucleosome does not alter the twist of the DNA significantly. A lower estimate of the linking number reduction associated with a two-turn nucleosome was also derived, based on an analysis of recent data obtained upon treatment of reconstituted minichromosomes with gyrase. The value, 1.6 turns, set a lower limit of 10.44 bp per turn for the twist of nucleosomal DNA, in agreement with the above estimate.(ABSTRACT TRUNCATED AT 400 WORDS)

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.