A physiological role for DNA supercoiling in the anaerobic regulation of colicin gene expression

Summary The mechanism of anaerobic regulation of synthesis of colicins E1, E2, E3, K and D was studied. It was found that anaerobiosis significantly increases expression of the genes for colicins E1, E2, E3, K, and D. Experiments with novobiocin (a DNA gyrase inhibitor) showed that colicin synthesis in minicells and derepressed colicin synthesis in cells are dramatically reduced by relaxation of DNA supercoiling. A good correlation was observed between the levels of colicin synthesis and plasmid DNA supercoiling and the degree of aeration of the cultures. Thus, the regulation of colicin gene expression in response to a change in aeration appears to be mediated by environmentally induced variations in DNA supercoiling.     

Potassium ions and changes in bacterial DNA supercoiling under osmotic stress

Escherichia coli transiently increases both the [ATP]/[ADP] ratio and the negative supercoiling of plasmid DNA when it is shifted to high osmolarity. Here we report that a mutant lacking all saturable K+ transport systems increases the negative supercoiling of the plasmid DNA under upshock but cannot further relax DNA. The mutant dnaK756 behaves like the K+ transport mutant.     

Differential influence of DNA supercoiling on in vivo strength of promoters varying in structure and organisation in E. coli

DNA supercoiling is known to influence promoter activity in vitro and in vivo in a promoter-dependent manner in prokaryotes. In order to investigate how topology may influence promoter function, we have studied two kinds of promoter variants, (i) where only the spacer region is altered, and (ii) where the same promoter is tandemly repeated in either the same or opposite orientation. These promoters respond very differently to alterations in DNA supercoiling, suggesting that the overall structure of the promoter and its context contribute to the differential response to alterations in supercoiling in vivo.     

Altered topoisomerase activities may be involved in the regulation of DNA supercoiling in aerobic-anaerobic transitions inEscherichia coli

This study uncovers a new mechanism of regulation of DNA supercoiling operativein vivo upon an aerobic-anaerobic transition inEscherichia coli. Exponentially growing aerobic batch cultures were subjected to a shift to anaerobic conditions. The ratio [ATP]/[ADP] remained essentially constant at 8.5 in the aerobic culture and after a transition to anaerobiosis while DNA supercoiling increased noticeably upon anaerobiosis. This result indicated that the mechanism of regulation of DNA supercoiling by the [ATP]/[ADP] ratio was not operative. The increase in DNA supercoiling was followed by a large decrease in the DNA-relaxing activity of topoisomerase I while gyrase activity remained relatively constant. This decrease in the activity of topoisomerase I is likely to be responsible for the increase in DNA supercoiling.Abbreviations TPE Tris-phosphate-EDTA buffer - TBE Tris-borate-EDTA buffer     

Buckling of adaptive elastic bone-plate: theoretical and numerical investigation

During day-to-day activities, many bones in the axial and appendicular skeleton are subjected to repetitive, cyclic loading that often results directly in an increased risk of bone fracture. In clinical orthopedics, trabecular fatigue fractures are observed as compressive stress fractures in the proximal femur, vertebrae, calcaneus and tibia, that are often preceded by buckling and bending of microstructural elements (Müller et al. in J Biomechanics 31:150 1998; Gibson in J Biomechanics 18:317-328 1985; Gibson and Ashby in Cellular solids 1997; Lotz et al. in Osteoporos Int 5:252-261 1995; Carter and Hayes in Science 194:1174-1176 1976). However, the relative importance of bone density and architecture in the etiology of these fractures are poorly understood and consequently not investigated from a biomechanical point of view. In the present contribution, an attempt is made to formulate a bone-plate buckling theory using Cowin's concepts of adaptive elasticity (Cowin and Hegedus in J Elast 6:313-325 1976; Hegedus and Cowin J Elast 6:337-352 1976). In particular, the buckling problem of a Kirchhoff-Love bone plate is investigated numerically by using the finite difference method and an iterative solving approach (Chen in Comput Methods Appl Mech Eng 167:91-99 1998; Hildebland in Introduction to numerical analysis 1974; Richtmyer and Morton in Difference methods for initial-value problems 1967).     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.     

Dynamics of DNA loop capture by the SfiI restriction endonuclease on supercoiled and relaxed DNA

The SfiI endonuclease is a prototype for DNA looping. It binds two copies of its recognition sequence and, if Mg(2+) is present, cuts both concertedly. Looping was examined here on supercoiled and relaxed forms of a 5.5 kb plasmid with three SfiI sites: sites 1 and 2 were separated by 0.4 kb, and sites 2 and 3 by 2.0 kb. SfiI converted this plasmid directly to the products cut at all three sites, though DNA species cleaved at one or two sites were formed transiently during a burst phase. The burst revealed three sets of doubly cut products, corresponding to the three possible pairings of sites. The equilibrium distribution between the different loops was evaluated from the burst phases of reactions initiated by adding MgCl(2) to SfiI bound to the plasmid. The short loop was favored over the longer loops, particularly on supercoiled DNA. The relative rates for loop capture were assessed after adding SfiI to solutions containing the plasmid and MgCl(2). On both supercoiled and relaxed DNA, the rate of loop capture across 0.4 kb was only marginally faster than over 2.0 kb or 2.4 kb. The relative strengths and rates of looping were compared to computer simulations of conformational fluctuations in DNA. The simulations concurred broadly with the experimental data, though they predicted that increasing site separations should cause a shallower decline in the equilibrium constants than was observed but a slightly steeper decline in the rates for loop capture. Possible reasons for these discrepancies are discussed.     

The ATP-operated clamp of human DNA topoisomerase IIalpha: hyperstimulation of ATPase by "piggy-back" binding

We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction.     

Electrostatic map of T7 DNA: comparative analysis of functional and electrostatic properties of T7 RNA polymerase-specific promoters

The entire T7 bacteriophage genome contains 39937 base pairs (Database NCBI RefSeq N1001604). Here, electrostatic potential distribution around double helical T7 DNA was calculated by Coulomb method using the computer program of Sorokin A.A. (lptolik@gmail.com). Electrostatic profiles of 17 promoters recognized by T7 phage-specific RNA polymerase were analyzed. It was shown that electrostatic profiles of all T7 RNA polymerase-specific promoters can be characterized by distinctive motifs which are specific for each promoter class. Comparative analysis of electrostatic profiles of native T7 promoters of different classes demonstrates that T7 RNA polymerase can differentiate them due to their electrostatic features.     

The structure of the sleeping genome: Implications of sperm DNA organization for somatic cells

The tertiary structure of the DNA that makes up the eukaryotic genome is remarkably plastic, taking many different forms in response to the different needs of the cell. During the cell cycle of one cell, the DNA is replicated, reorganized into mitotic chromosomes, and decondensed into interphase chromatin. Within one cell at any given point in time, the chromatin is divided into hetero- and euchromatin reflecting active and inactive states of the DNA. This organization varies within one organism since different parts of the genome are active in different cell types. This article focuses on the most dramatic cell-type-specific DNA organization, that found in spermatozoa, in which the entire genome is reorganized into an inactive state that is more highly condensed than mitotic chromosomes. This unique example of eukaryotic DNA organization offers some interesting clues to the still unanswered questions about the role that the three-dimensional packaging of DNA plays in its function. © 1994 Wiley-Liss, Inc.     

Analysis of chemical and enzymatic cleavage frequencies in supercoiled DNA

Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively supercoiled plasmid. Our results support the presence of a stem-loop structure in the expected location and suggest that the helical geometry of the cruciform stem differs from that of the normal duplex form.     

Heterogeneity in the level of ampicillin resistance conferred by pBR322 derivatives with different DNA supercoiling

Summary Cloning of an EcoRI restriction fragment, containing the 900 bp -terminal sequence of transposon Tn1000, into pBR322, resulted in two plasmids, pICV63 and pICV64, which differed in the orientation of the cloned fragment within the replicon and in the level of ampicillin resistance conferred on the host cell. The DNAs of these plasmids differ in superhelicity and we suggest that a change in supercoiling of pICV63 DNA leads to this plasmid conferring resistance to only low levels of ampicillin, probably by reducing the expression of the bla gene. This hypothesis is supported by the fact that topA or supX mutations, which abolish topoisomerase I, reduce still further the level of resistance to ampicillin of pICV63-containing cells, whereas the gyrB226 compensatory mutation renders these cells more ampicillin resistant. Plasmid pICV63, therefore, enables mutant alleles of genes governing DNA topology to be recognized.     

Structure,function and DNA composition of Saccharomyces cerevisiae chromatin loops

Recent localization of cohesin association regions along the yeast chromatin fibre suggests that compositional variability of DNA in yeast is related to the function and organization of the chromosomal loops. The bases of the loops, where the chromatin fibre is attached to the chromosomal axis, are AT-rich, bind cohesin, and are flanked by genes transcribed convergently. The hotspots of meiotic recombination are mainly found in the GC-rich parts of the loops, ‘external’ with respect to the chromosomal axis, frequently in the vicinity of the promoters of divergently transcribed genes. There are two possible reasons why the regions of the hotspots of recombination were enriched in GC content during evolution. One is a biased repair of recombination intermediates, and the second is a selective advantage due to an increased chromatin accessibility, which may have the carriers of GC-enriched alleles over the carriers of AT-rich alleles.