Transfer of nucleosomes from parental to replicated chromatin.

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.     

Disruption of the nucleosomes at the replication fork.

The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt-treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T-antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross-linking of the DNA and by micrococcal nuclease digestion. A 5- and 10-fold excess of protein-free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.     

Comparative effect of microwaves and boiling on the denaturation of DNA

The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.     

Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli.

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.     

Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.

Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.     

Effects of excluded volume upon protein stability in covalently cross-linked proteins with variable linker lengths

To explore the effects of molecular crowding and excluded volume upon protein stability, we used a series of cross-linking reagents with nine different single-cysteine mutants of staphylococcal nuclease to make covalently linked dimers. These cross-linkers ranged in length from 10.5 to 21.3 A, compelling separations which would normally be found only in the most concentrated protein solutions. The stabilities of the dimeric proteins and monomeric controls were determined by guanidine hydrochloride and thermal denaturation. Dimers with short linkers tend to exhibit pronounced three-state denaturation behavior, as opposed to the two-state behavior of the monomeric controls. Increasing linker length leads to less pronounced three-state behavior. The three-state behavior is interpreted in a three-state model where cross-linked native protein dimer, N-N, interconverts in a two-state transition with a dimer where one protein subunit is denatured, N-D. The remaining native protein in turn can denature in another two-state transition to a state, D-D, in which both tethered proteins are denatured. Three-state behavior is best explained by excluded volume effects in the denatured state. For many dimers, linkers longer than 17 A removed most three-state character. This sets a limit on the flexibility and size of the denatured state. Notably, in contradiction to theoretical predictions, these cross-linked dimers were not stabilized. The failure of these predictions is possibly due to neglect of the alteration in hydrophobic exposure that accompanies any significant reduction in the conformational space of the denatured state.     

DNA stability at temperatures typical for hyperthermophiles.

We have studied the fate of covalently-closed circular DNA in the temperature range from 95 to 107 degrees C. Supercoiled plasmid was not denatured up to the highest temperature tested. However, it was progressively transformed into open DNA by cleavage and then denatured. Thermodegradation was not dependent on the DNA supercoiling density. In particular, DNA made positively supercoiled by an archaeal reverse gyrase was not more resistant to depurination and thermodegradation than negatively supercoiled DNA. Thermodegradation was similar in aerobic or anaerobic conditions but strongly reduced in the presence of physiological concentrations of K+ or Mg2+. These results indicate that the major problem faced by covalently closed DNA in hyperthermophilic conditions is not thermodenaturation, but thermodegradation, and that intracellular salt concentration is important for stability of DNA primary structure. Our data suggest that reverse gyrase is not directly required to protect DNA against thermodegradation or thermodenaturation.     

Osmium-induced alteration in DNA structure

In the presence of pyridine and other ligands osmium tetroxide binds covalently to pyrimidine bases in DNA. Properties of osmium-modified native and denatured calf thymus DNA, and plasmid Co1E1 DNA were investigated by means of differential pulse polarography, absorption spectrophotometry, circular dichroism, agarose gel electrophoresis, and nuclease S1 digestion. A great difference in the reaction kinetics of native and denatured DNAs with osmium, pyridine was observed. On the ground of the slow stepwise reaction kinetics of native DNA in the initial stage of its modification by osmium it has been suggested that the primary reaction sites do not include bases contained in the intact double helix. Osmium binding to sporadic primary reaction sites (represented e.g. by bases in the vicinity of a single-strand break) in native calf thymus DNA resulted in local changes in DNA conformation limited to a close neighbourhood of the binding site. At higher osmium/nucleotide ratios disordering of the DNA structure over a region extending beyond the immediate binding site was observed. With denatured DNA the same type of structure disordering was detected already in the initial stage of the reaction at osmium/nucleotide ratios as low as 0.01. Osmium binding to the supercoiled Co1E1 DNA resulted in its relaxation without nicking and it increased its sensitivity to linearization by cleavage with nuclease S1. The behaviour of Co1E1 DNA has been explained by the formation of a denatured region in the molecule (accompanied by a coupled loss of duplex and superhelical turns). It has been suggested that osmium can be used to label and to visualize distorted regions in the DNA double helix.     

Transforming activity of phage T4r+ DNA, treated with ultraviolet light, nitrous acid, hydroxylamine and visible light in the presence of methylene blue]

The efficiency of phages T4rIIB-638v+ and T4rIIB-638v- transformation by native and denatured DNA treated with UV, nitrous acid, hydroxylamine and visible light in the presence of methylene blue is studied. A greater transformation efficiency of UV-irradiated T4r+ phage native and denatured DNA was observed in the v+ recipient as compared with v- recipients. Denatured donor DNA treated with nitrous acid has higher transformation activity in spheroplasts infected with T4v+ phage than in those infected with T4v- phage. Native donor DNA, treated with methylene blue and visible light-irradiated, developed a decrease of the transformation activity in T4v- phage-infected spheroplasts as compared with T4v+ phage-infected spheroplasts. Hydroxylamine treatment of native and denatured donor DNA did not reveal any differences in the transforming activity for v+ and v- recipients. Denatured donor DNA was more resistant to the effect of hydroxylamine than native DNA.     

Heat denaturation of human orosomucoid in water/methanol mixtures

Heat denaturation of orosomucoid in solutions of methanol concentrations ranging from 0 to 70% (v/v) has been studied by using circular dichroism, intrinsic protein fluorescence and thermal difference absorption spectroscopy. Regardless of its high saccharide content (40%), the highly cooperative denaturation transition of orosomucoid is fully reversible in neutral water solution. A two-state model has been successfully applied; the numerical analysis results in thermodynamical parameter values that are in close agreement with previously reported experimental data from calorimetric measurements. However, in solutions containing even minute concentrations of methanol (5%) the heat denaturation is irreversible. After cooling of the denatured protein the refolded molecules exhibit a higher α-helical content than the native one. Possibilities of methanol interaction with native and denatured protein molecule are discussed.     

Replication of kinetoplast DNA of Crithidia acanthocephali. I. Density shift experiments using deuterium oxide

The protozoan Crithidia acanthocephali contains, within a modified region of a mitochondrion, a mass of DNA known as kinetoplast DNA (kDNA). This DNA consists mainly of an association of approximately 27,000 covalently closed 0.8-mum circular molecules which are apparently held together in a definite ordered manner by topological interlocking. After culturing of C. acanthocephali cells for 25 generations in medium containing 75% deuterium oxide, both nuclear DNA (rhonative, nondeuterated=1.717 g/cm3) and kDNA (rhonative, nondeuterated=1.702 g/cm3) increased in buoyant density by 0.012 g/cm3. The replication of the two DNAs was studied by cesium chloride buoyant density analysis of DNAs from exponentially growing cells taken at 1.0, 1.4, 2.0, 3.0, and 4.0 cell doublings after transfer of cells from D2O- containing medium into medium containing only normal water. The results obtained from analysis of both native and denatured nuclear DNAs indicate that this DNA replicates semiconservatively. From an analysis of intact associations of kDNA, it appears that this DNA doubles once per generation and that the newly synthesized DNA does not segregate from parental DNA. Fractions of covalently closed single circular molecules and of open circular and unit length linear molecules were obtained from associations of kDNA by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium gradient centrifugation. Buoyant density profiles obtained from these fractions indicate that: (a) doubling of the kDNA results from the replication of each circular molecule rather than from repeated replication of a small fraction of the circular molecules; (b) replication of kDNA is semiconservative rather than conservative, but there is recombination between the circles at an undefined time during the cell cycle.     

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.     

Early melting of supercoiled DNA.

Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.     

The effect of supercoil and temperature on the recognition of palindromic and non-palindromic regions in phi X174 replicative form DNA by S1 and Bal31

The effect of supercoil and temperature on the topology of phi X174 replicative form (RF) DNA was studied using single-strand specific endonucleases S1 and Bal31 as probes for cruciform extrusion and other structural perturbations of the B-helix. Both enzymes were found to recognize specifically and reproducibly over 30 sites, most of which were cleaved by both enzymes independent of the superhelicity of the genome. A negative superhelical density exceeding 0.06 stabilized a transition in the DNA conformation that generated several new cleavage sites for Bal31. The underlying structures appeared to be only transiently stable and were lost from in vitro supercoiled DNA during brief incubation at 65 degrees C. They were generally absent from in vivo supercoiled RF DNA of equal superhelicity as a consequence of the extraction and storage procedure. Mapping of the cleavage sites suggested that they were preferentially located near the beginnings and ends of genes and that the structural basis for at least some of them was the extrusion of relatively small palindromes into the cruciform state. Insertion of a short synthetic palindromic sequence into the phi X174 genome generated a supercoil-dependent, temperature-sensitive secondary structure that was cleaved in the Bal31 but not the S1 reaction, further supporting the hypothesis that even small cruciforms with stem size of 7 or less base pairs may be transiently stable. Subjecting supercoiled RF DNA to the typical S1 reaction conditions induced a topological shift that diminished all but one of the supercoil-induced Bal31 recognition sites and promoted the formation of one major new site.     

Gel electrophoretic technique for separating crosslinked RNAs. Application to improved electron microscopic analysis of psoralen crosslinked 16 S ribosomal RNA

We have developed a gel electrophoresis technique for separating crosslinked RNA molecules into a series of discrete fractions. The gel used is polyacrylamide made in formamide and low salt designed to denature the RNA during electrophoresis. The mobility depends upon the position of crosslinking within each molecule, as demonstrated by electron microscopy of RNA eluted from the gel. In general, molecules with large loops electrophorese more slowly than molecules with small loops or uncrosslinked molecules. We have used this technique to re-examine the psoralen crosslinking pattern of Escherichia coli 16 S ribosomal RNA in inactivated 30 S ribosomal subunits. To determine the correct orientation of each type of crosslink, we have covalently attached DNA restriction fragments to the RNA so that the polarity of the RNA in the microscope would be known. Our previous major conclusions are confirmed: the predominant long-distance crosslink detected by gel electrophoresis involves a residue close to the 3′ end and a residue approximately 600 nucleotides away: the formamide/polyacrylamide gel is able to separate two closely spaced 1100-nucleotide interactions beginning close to the 3′ end, which were reported as one interaction before: and an interaction joining the ends is detected as before. However, one low-frequency crosslinked interaction, between positions 950 and 1400, and possibly another low-frequency interaction, between positions 550 and 870, are determined to be in the opposite polarity to that described previously.     

New genes with old modus operandi. The connection between supercoiling and partitioning of DNA in Escherichia coli

The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis. In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell. As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork. The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity. Thus, genes involved in chromosome topology, once set aside as ‘red herrings’ in the search for ‘true’ partition functions, are again recognized as being important for chromosome partitioning in E. coli.     

A DNA endonuclease isolated from yeast nuclear extract.

We have isolated and partially purified a DNA endonuclease from nuclei of the yeast Saccharomyces cerevisiae. Although purified on the basis of its ability to degrade denatured DNA, the enzyme can also attack native DNA. Denatured oligonucleotide products of the enzyme are sensitive to venom phosphodiesterase (EC3.1.4.1.) but not to bovine spleen phosphodiesterase (EC3.1.4.18). The enzyme has an estimated molecular weight of 6.6--7.5 X 10(4), more than twice as large as the endonucleases involved in DNA repair in Escherichia coli. When analyzed on glycerol gradients, the endonuclease sedimented as a single activity against both denatured DNA and closed circular DNA duplexes. The enzyme showed a 10-fold preference for denatured over native T7 DNA substrate, and appears to produce random nicks in a supercoiled replicative form of phiX174 DNA (RFI) with no discernable preference for the unpaired bases in the supercoiled duplex. The endonuclease appears to be distinct from the yeast endonucleases previously described.     

HEAT DENATURATION STUDIES OF RAT LIVER MITOCHONDRIAL DNA : A Denaturation Map and Changes in Molecular Configurations

The effect of progressive denaturation of open circular molecules (component II) and supercoiled covalently closed circular molecules (component I) of rat liver mitochondrial DNA has been followed by heating in the presence of formaldehyde and examination in the electron microscope. After heating at 49°C, two, three, or four regions of strand separation were visible in 25% of the component II molecules. Comparisons of the patterns of distribution of these regions in individual molecules indicated that they occurred at at least three specific positions around the molecule. Also, these regions, which were assumed to be rich in adenine and thymine, were within a segment which was less than 50% of the length of the molecule. After heating at 50°C, up to 14 regions of strand separation were observed, but when comparisons were made no clear groupings were found. At 51°C, component II molecules were completely separated into a single-stranded circle and a single-stranded linear piece of similar length. Strand separation was accompanied by shortening of the molecule. At 70°C, single-stranded circles had a mean length of 2.7 µ, compared with 5.0 µ for native molecules. Progressive heating of component I molecules resulted first in conversion to an open circle (I') and then to a second supercoiled form (I'). Visualization of further denaturation products of component I was prevented by crosslinking of the molecule by formaldehyde at high temperatures.