Isolation,characterization, and structure of the folded interphase genome of Drosophila melanogaster

The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% Nonidet P40. The nonviscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranucleosome histones: H2A, H2B, H3, and H4).The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs—that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26°C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres.Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA.The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA-DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360° left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.     

Properties of the relaxation complex of supercoiled deoxyribonucleic acid and protein of R plasmid NR1 in Escherichia coli.

Some properties of the supercoiled deoxyribonucleic acid (DNA)-protein relaxation complex of the R plasmid NR1, which contains more than one origin for DNA replication, were examined. The percentage of complexed NR1 molecules that can be converted to the relaxed (nicked) form appeared to be unaffected by the conditions under which the host cells were cultured. However, the percentage of supercoiled NR1 DNA that can be relaxed was highly dependent on the method used to prepare the DNA and the agents used to induce relaxation. Our data suggest that 100% of NR1 molecules may exist in situ as DNA-protein relaxation complexes. An RTF-Tc segregant of NR1, which has deleted the r-determinants component of the NR1 and therefore does not contain the two origins of replication located in the r-determinants, has indistinguishable relaxation properties in comparison with NR1 itself.     

Relationship of R6K replicating forms to the folded chromosome of Escherichia coli.

An examination of the relationship of both nonreplicating and replicating forms of R6K plasmid DNA to the Escherichia coli folded chromosome showed that both forms cosediment with the chromosome in neutral sucrose gradients. Approximately 20% of the nonreplicatin molecules was found as freely sedimenting forms when the folded-configuration of the chromosomes was preserved. However, under the same conditions negligible amounts of the replicating forms were found as freely sedimenting molecules. Thus, it is concluded that the replicating forms, when compared with nonreplicating molecules, are preferentially associated with the folded chromosomal structure. Exposure of the folded chromosomal structure to RNase resulted in an unfolding of the chromosome and a concomitant increase in the amount of freely sedimenting replicating and nonreplicating forms of R6K DNA. Analyses of the single-stranded length of RNase-released nascent molecules suggest that they replicate in continuous association with the folded chromsome complex. Nonenzymatic unfolding of the chromosomes by progressively lowering the sodium ion concentration during lysis resulted in a progressive increase in the release of nonreplicating molecules. Replicating molecules wer not released by unfolding the chromosome in this fashion.     

The helical repeat of underwound DNA in solution.

Closed circular DNA was relaxed with a topoisomerase in the presence of varying concentrations of the intercalating dye, ethidium bromide, to create underwound, planar DNA rings. We directly determined the helical repeat of these DNA molecules by the Gaussian center method and found that it varied as a simple predicted function of the degree of underwinding and the helical repeat of relaxed, dye-free DNA. We discuss these results in light of a recent mathematical treatment of DNA structure which predicts that the helical repeat of supercoiled DNA molecules in solution obeys the same function.     

Stable DNA-protein complexes in eukaryotic chromatin

Demembranized sperm and somatic nuclei of mammalian origin were extracted with high salt/urea/2-mercaptoethanol, treated with detergents and purified in CsCl density gradients to isolate DNA. Under these conditions a protein component still remained bound to DNA. This stable DNA-protein complex could be reduced to an oligodeoxynucleotide-peptide complex by extensive sequential digestions with DNase I and Pronase E. Chemical and enzymatic treatments of this complex indicated the presence of a phosphoester bond between DNA and a hydroxyamino acid. Two-dimensional tryptic peptide mapping revealed a remarkable similarity among the covalently linked protein components in all types of chromatin studied. These maps differed from the maps of mammalian topoisomerases I and II.     

A protein covalently bound to ColE1 DNA

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColE1 DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane-treated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColE1 DNA via a phosphodiester bond between dC and the serine residue.     

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.     

Supercoils in human DNA.

The three-dimensional structure of a double-stranded DNA molecule may be described by distinguishing the helical turns of the DNA duplex from any superhelical turns that might be superimposed upon the duplex turns. There are characteristic changes in the hydrodynamic properties of superhelical DNA molecules when they interact with intercalating agents. The hydrodynamic properties of nuclear structures released by gently lysing human cells are changed by intercalating agents in this characteristic manner. The characteristic changes are abolished by irradiating the cells with gamma-rays but may be restored by incubating the cells at 37 degrees C after irradiation. These results are interpreted as showing that human DNA is supercoiled. A model for the structure of the chromosome is suggested.     

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.     

A specific inhibitor of type I DNA-topoisomerase of Trypanosoma cruzi: dimethyl-hydroxy-ellipticinium

2-6 dimethyl-9-hydroxyellipticinium inhibited the relaxation of supercoiled DNA by the type I topoisomerase of T. cruzi. Since DNA relaxed in the presence of an intercalating drug prior to electrophoresis became supercoiled when the ligand was removed, we analysed the topoisomerisation in gels containing another ligand, chloroquine. The inhibition which is reported here, concerning a type I topoisomerase, is of an exceptional efficiency.     

The Xenopus laevis mitochondrial protein mtDBP-C cooperatively folds the DNA in vitro.

The binding of the Xenopus laevis mitochondrial protein mtDBP-C to DNA was studied by equilibrium density banding, agarose gel electrophoresis and electron microscopy. The results obtained show that the mtDBP-C binds cooperatively to DNA irrespective of whether the DNA is supercoiled, relaxed or linear and it induces the formation of superhelical turns locally leading to the formation of a highly folded structure. It appears that this protein could be involved in the compaction of DNA in the mitochondrial nucleoid.     

Role of nonhistone proteins in metaphase chromosome structure

In this paper, we show that HeLa metaphase chromosomes still possess a highly organized structure retaining the familiar metaphase morphology following removal of virtually all the histones and most of the nonhistone proteins. The structure is stabilized by a relatively small number of nonhistones, which we call scaffolding proteins.These results are based on a method which allows the removal of the histones, and most of the nonhistone proteins, by competition with polyanions such as dextran sulfate and heparin.The histone-depleted chromosomes sediment in sucrose gradients as a broad peak between 4000 to 7000S. These structures are dissociated by mild trypsin or chymotrypsin treatment, or by 4 M urea, but are stable in 2 M NaCl and insensitive to treatment with RNAase A. The histone-depleted chromosomes have a DNA to protein ratio of about 6:1; gel electrophoresis reveals the presence of about 30 nonhistone proteins and the virtual absence of histones. These experiments suggest that nonhistone proteins exist in metaphase chromosomes which maintain the DNA chain in a highly folded conformation.Structural studies support this conclusion. Analysis by fluorescence microscopy of histone-depleted chromosomes stained with ethidium bromide shows that each chromatid is still paired with its sister chromatid, and consists of a central structure surrounded by a halo of DNA. The length of the central structure in each chromatid is about 2–3 times longer than the chromatid length in the original chromosome.     

Inhibition of the reactions catalysed by a type I topoisomerase and catenating enzyme of Trypanosoma cruzi by DNA-intercalating drugs. Preferential inhibition of the catenating reaction

A catenating enzyme and a type I topoisomerase were purified from Trypanosoma cruzi. We investigated the inhibitory effect of DNA-intercalating drugs on topoisomerisations catalysed by these enzymes. Inhibition of catenation was detected by electrophoretic analysis in neutral agarose gels. However, the inhibition of relaxation was not readily detectable in these gels since supercoiled DNA, which was relaxed in the presence of an intercalating drug, returned to a supercoiled state when the drug was removed. Thus electrophoretic analyses were made in gels containing chloroquine so that unreacted DNA could be distinguished from DNA relaxed by the enzyme. The results show that the catenation was more sensitive to DNA-intercalating drugs than the relaxation.     

Properties of Nucleoprotein Complexes Containing Replicating Polyoma DNA

Short-lived nucleoprotein complexes (r-py complex) containing replicating polyoma DNA were isolated from infected cells after lysis with Triton X-100. The Triton lysing procedure of Green, Miller, and Hendler (1971) releases most complexes containing supercoiled viral DNA (py complex) from nuclei, but liberates only a portion of r-py complexes. r-py Complexes are associated more strongly with nuclear sites but can be extracted by prolonged incubation of nuclei in lysing solution. Complexes containing replicating polyoma DNA appear to be precursors to stable complexes containing supercoiled DNA. Sedimentation and buoyant density studies indicate that protein is bound to both r-py complexes and py complexes at a ratio of protein to DNA of about 1 to 2/1. Both types of complexes sediment as if the viral DNA is more compact than free DNA and both undergo major reversible configurational changes with increased salt concentration. Changes resulting from enzymatic and chemical treatment indicate that there may be two or more protein components in both r-py complex and py complex. One component is digested by Pronase and trypsin while another is resistant to the enzymes but released by deoxycholate. The abundance and similarity in chemical and physical properties of protein bound to all forms of polyoma DNA suggest that part of the protein molecules may serve in a structural capacity.     

Characterization of supercoiled nucleoprotein complexes released from detergent-treated vaccinia virions.

Treatment of vaccinia virions with 1% sodium dodecyl sulfate in the absence of reducing agents resulted in the release of subviral particles termed "subnucleoids," which contained viral DNA in combination with four polypeptides with molecular weights of 90,000, 68,000, 58,000 and 10,000. Biochemical and electron microscopic studies showed that viral DNA in combination with these polypeptides was maintained in a superhelical configuration. When subnucleoids were "fixed" with glutaraldehyde and formaldehyde and then examined by electron microscopy, spherical particles were observed, in which the supercoiled DNA was folded into globular structures that were 20 to 60 nm in diameter and were interconnected by DNA-protein fibers resembling the nucleosome structures described for eucaryotic chromatin.     

Absence of anthracycline-induced degradation of nuclear DNA in Ehrlich ascites tumour cells

The in vivo effects of anthracycline antibiotics on the integrity of Ehrlich ascites tumour cell DNA have been studied by sedimentation analysis of nuclear structures containing superhelical DNA in neutral sucrose gradients. These fast-sedimenting protein-DNA complexes may be released by gently lysing cells in solution containing non-ionic detergents and high NaCl concentrations (1.95 M). The supercoiled structure of DNA in these protein-DNA complexes is suggested by the characteristic sedimentation in the presence of intercalating agents. Apparently, no DNA damage could be detected in Ehrlich cells from 7-day-old tumours within 3 h after various doses of daunomycin (0.5–10 mg/kg of body wt.) were administered i.p. to mice. Sedimentation anomalies could not be observed even 15 or 30 h after administration of therapeutic doses of daunomycin or adriamycin. In contrast, at 30 min after administration to mice, therapeutic doses of bleomycin (2–8 mg/kg) caused extensive fragmentation of tumour cell DNA, which could be monitored as slowly sedimenting DNA structures (compared with the control). Similarly, DNA damage could be induced by procarbazine at therapeutic doses. Exposure to bleomycin or procarbazine abolished the characteristic biphasic response to ethidium bromide. The absence of anthra-cycline-induced degradation of Ehrlich ascites tumour cell DNA is apparently in contrast with the DNA damage observed in L1210 tumour cells. These observations suggest that DNA damage is not a necessary condition for antitumour activity.     

A compact form of rat liver mitochondrial DNA stabilized by bound proteins.

A highly folded, rapidly sedimenting form of rat liver mitochondrial DNA has been released from the organelles wiht BRIJ 58 and sodium deoxycholate in the presence of 0.5 M NaCl and isolated by sedimentation velocity in sucrose gradients. Under these conditions a majority of the mitochondrial DNA labeled in vitro sedimented beyond 39 S, the sedimentation coefficient of a highly purified mitochondrial DNA supercoil, and appeared as a stable, heterogeneous population of species ranging in s values between 42 S and about 70 S. Under formamide-spreading conditions most of the rapidly sedimenting forms appeared in the electron microscope as single genome length rosettes constrained at the center in a dense core. Except for an occasional D-loop, no extraordinary structural features were evident along the smooth loops projecting radially from the central core. In sucrose gradients containing various amounts of ethidium bromide, the sedimentation velocity of the folded DNA changed in a biphasic fashion in response to increasing amounts of dye. At a dye concentration of 0.5 microgram per ml the DNA species present reached s value minima, but two major peaks sedimenting at 32 S and 42 S were present at this point. Thus, although these species were similar in superhelix density, there appeared to be additional constraints superimposed upon their tertiary structure that folded these forms to differing degrees of compactness. Direct chemical analyses showed that proteins were bound to the folded DNA at a protein to DNA ratio of about 0.3. Separation of the bound proteins on SDS-polyacrylamide gels revealed an array of proteins ranging in molecular weight between 11,000 and 150,000. Several of the lower molecular weight proteins co-migrated with proteins from the inner mitochondrial membrane, but the major DNA-bound band (Mr = 58,000) was undetectable among the proteins from any other submitchondrial fraction. Digestion of the compact DNA structure with proteinase K under various conditions indicated that the DNA was maintained in the compact conformation by the tightly bound proteins and that the portions of these proteins directly involved in stabilizing the folded DNA were proteinase insensitive unless digestion was carried out in the presence of a disulfide reductant at elevated temperatures.     

Analysis of DNA-protein complexes induced by chemical carcinogens.

DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.     

DNA bending,compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus

Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea Sulfolobus. Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments.