Repair of thermal damage to the Escherichia coli nucleoid.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.     

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.     

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 degrees C and 45 degrees C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 degrees C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells (45 degrees C for 15 min) were incubated at 37 degrees C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 degrees C (step-down heating; SDH) a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks.     

A note on the repair of the Escherichia coli nucleoid structure after heat shock

The repair of the Escherichia coli nucleoid structure after heat shock (50 degrees C, 5 min) was studied. After heat shock the repair process did not include the association of the nucleoid to protein structures as is the case after more severe heat treatments resulting in cell death or inactivation.     

Changes in nucleoid viscosity following X-irradiation of rat thymic and splenic cells in vitro

Summary Unscheduled DNA synthesis (UDS) suggested a higher DNA repair capacity of X-irradiated rat thymic (T) cells when compared to splenic (S) cells (Tempel 1980). In the present investigations, damage and repair of DNA supercoiling was measured in T- and S-cells following X-irradiation in vitro by using the nucleoid sedimentation technique and a simplified low-shearing viscometric test. - X-irradiation resulted in a dose (0.6–19.2 Gy) - dependent reduction in sedimentation and viscosity of nucleoids. Within a post-irradiation period of 30–45 min after a challenge dose of 19.2 Gy, DNA repair was accompanied by an increase in nucleoid sedimentation and viscosity in T-cells by about 60 and 300, in S-cells by almost 40 and 100%, resp. The increase in nucleoid viscosity within a 30 min repair period could be reduced in a concentration-dependent manner by DNA polymerase - inhibitors and proteinase K. - The higher DNA repair capacity of T-cells as reflected by UDS is confirmed therefore by the nucleoid characteristics. Apart from this suggestion, measuring nucleoid viscosity may be considered as a sensitive, simple and rapid device to detect radiation-induced DNA supercoiling phenomena.     

Competence growth factors can cause modification in higher-order chromatin structure in mouse embryo 3T3 fibroblasts

The authors compared sedimentation rates of nucleoids from mouse embryo 3T3 fibroblasts cultured in the presence or absence of different cell growth factors. The results clearly showed that rapidly sedimenting nucleoids are obtained only when cells are supplied with any of the following competence growth factors: platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or the product of the oncogene v-sis (a peptide homologous to PDGF). The tumor promoter phorbol 12-myristate 13-acetate, an activator of protein kinase C and a partial mitogen, shares this property with the competence growth factors. Removal of these factors from the medium causes cells to enter Go and nucleoids to sediment at a slower rate. Protein synthesis is required for growth factor induction of change in nucleoid sedimentation, but inhibition of either DNA synthesis or DNA repair does not antagonize the effect of growth factors. Titration of nucleoids with ethidium bromide indicates that one possible mechanism for the nucleoid change is the unwinding of DNA in supercoiled loops. The results indicated that the nucleoid change constitutes a cell response to competence factors that might have an important role in cell proliferation.     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

A note on the repair of the Escherichia coli nucleoid structure after heat shock

The repair of the Escherichia coli nucleoid structure after heat shock (50°C, 5 min) was studied. After heat shock the repair process did not include the association of the nucleoid to protein structures as is the case after more severe heat treatments resulting in cell death or inactivation.     

Delayed nucleoid segregation in Escherichia coli

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.     

Effect of adriamycin and hyperthermia on the sedimentation of nucleoids from L1210 cells

We report on the individual and combined effects of adriamycin (ADR) and hyperthermia (HYP) on the sedimentation behavior of L1210 mouse leukemia cell nucleoids in neutral sucrose gradients. Nucleoid sedimentation profiles obtained from cells incubated with ADR (1-10 microM; 30 min; 37 degrees C) exhibited an increased sedimentation rate associated with an increased protein content of these subnuclear units. Exposure of cells to HYP (1-3 h; 42 degrees C) produced similar results. Simultaneous exposure of L1210 cells to conditions of HYP and ADR which resulted in minimal changes in nucleoid sedimentation when used singly, produced an enhanced effect. A similar enhancement was observed with other intercalating antineoplastic agents believed to exert their effect, at least partially, via free radicals (daunorubicin, amsacrine, bisantrene, mitoxantrone). However, enhancement with HYP was not observed with (a) the classic intercalating agent, ethidium bromide; (b) non-intercalating DNA-breaking agents (bleomycin, lithocholic acid, etoposide); (c) inhibitors of poly (ADP-ribose) polymerase (m-methoxybenzamide, benzamide); or (d) non-intercalating antineoplastic agents capable of causing free radical formation (bleomycin). The results suggest that DNA intercalating agents capable of initiating free radical processes may show an enhanced toxicity with simultaneous HYP treatment, and that the nucleoid assay may be a means of screening agents with these biological properties for potential clinical usefulness in combination with HYP.     

Bacterial chromosome segregation: evidence for DNA gyrase involvement in decatenation

Nucleoids isolated from a temperature-sensitive gyrB mutant of E. coli, incubated at restrictive temperatures, exhibit increased sedimentation rates and an abnormal doublet or dumbbell-shaped morphology. Shifting cells from restrictive to permissive temperature prior to nucleoid isolation leads to decreases in the percentage of doublet nucleoids and in nucleoid sedimentation rates. When nucleoids isolated from mutant cells exposed to restrictive temperature are incubated with purified gyrase, the percentage of doublet nucleoids decreases as the total number of nucleoids increases. These results, together with the demonstrated ability of gyrase to decatenate small circular DNA molecules in vitro, suggest that gyrase participates in bacterial chromosome segregation through its decatenating activity.     

Mitomycin-C-induced changes in the nucleoid of Escherichia coli K12

The influence of low concentrations of mitomycin-C on the structure of the envelope-free nucleoid was studied in several strains of Escherichia coli K12. The wild-type strain AB1157 uvr+ rec+ and 3 mitomycin-C-sensitive derivatives carrying mutations in the uvrA, uvrB and recA genes, were used. Treatment of the control strain with mitomycin-C, 0.5 microgram/ml, followed by incubation in drug-free medium resulted in the formation of a transient fast-sedimenting nucleoid with a sedimentation coefficient of 2200 S. A fraction of 25% of the nucleoids had attained the normal sedimentation coefficient of 1570 S 3 h after removal of mitomycin-C. With the uvr- strains, mitomycin-C induced a slow, almost linear increase in the S value of the envelope-free nucleoid. In these cases the S value continued to increase during post-incubation and was 2050 S 3 h after removal of the drug. Post-incubation of recA- cells resulted in loss of supercoiling, decrease in S value of the nucleoid and degradation of DNA. Results obtained with phase-contrast and electron microscopy were in good agreement with the hydrodynamic data.     

Studies on the compaction of isolated nucleoids from Escherichia coli

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.     

The interaction of heat and radiation affecting the ability of nuclear DNA to undergo supercoiling changes

DNA damage (putatively strand breaks) from ionizing radiation inhibits the ability of intercalating dyes to induce right-handed supercoils in the DNA loops of HeLa nucleoids [Cook and Brazelle, J. Cell Sci. 22, 287-302 (1976); Roti Roti and Wright, Cytometry 8, 461-467 (1987)] while heat-induced changes in the nuclear matrix enhance this ability [Roti Roti and Painter, Radiat. Res. 89, 166-175 (1982)]. Since heat and radiation interact synergistically or additively on most cellular functions which they affect, the rewinding of DNA supercoils is unusual in that these agents alone affect it in an antagonistic manner. When HeLa cells were exposed to 45 degrees C for 30 min and immediately irradiated with 10 Gy of 137Cs gamma rays, the rewinding response was intermediate between that for cells which had been exposed to 10 Gy only and control. When repair of this damage was assayed in control cells, 97% of the initial damage had been repaired at 30 min postirradiation; at the same time only 10% of the initial damage had been repaired in the heat-shocked cells. This apparent dose reduction effect and the inhibition of repair were interpreted to indicate that heat-induced changes in nuclear structure were masking DNA damage from the assay and the repair system. These effects correlated with the amount of heat-induced excess protein associated with the nucleus and the nucleoid.     

Analysis of metal-induced DNA lesions and DNA-repair replication in mammalian cells

The potency of several metal compounds in causing lesions in DNA either directly or by exposure of intact cultured cells has been examined using the neutral conditions of nucleoid gradient sedimentation. HgCl2 was clearly the most potent inducer of single-strand breakage when added to isolated nucleoids or when nucleoids were prepared from cells treated with this compound. CaCrO4 , however, caused DNA-strand breaks in nucleoids isolated from cells treated with this agent but did not induce DNA strand breaks when added directly to nucleoids. Although less potent than HgCl2, NiCl2 also caused significant single strand breakage in isolated nucleoids or in nucleoids prepared from cells treated with this metal. Since strand breakage of DNA in intact cells may occur secondary to activation of DNA-dependent nucleases during repair replication, CsCl gradient density sedimentation was utilized to examine whether repair processes were induced by exposure of cells to NiCl2, HgCl2 and CaCrO4 . CaCrO4 and NiCl2 induced substantial DNA-repair activity at concentrations and exposure times where DNA lesions could not be detected whereas HgCl2 induced a 10-fold lower level of DNA-repair activity compared to CaCrO4 at optimal concentrations which again were below the concentrations of this metal that produced measurable DNA lesions. Both the induction of DNA-repair activity and DNA-strand breakage by these metals was concentration- and time-dependent. These results demonstrate some unique aspects of the interaction of HgCl2, NiCl2 and CaCrO4 with the DNA of intact cells and point to the possible important correlation of induction of DNA repair to carcinogenesis since nickel and chromate have clearly been implicated as carcinogens and induce considerable repair whereas HgCl2 is not considered a carcinogen and induces the least DNA repair despite its potency in producing DNA lesions.     

Absence of swiveling at sites of intercalator-induced protein-associated deoxyribonucleic acid strand breaks in mammalian cell nucleoids

The sedimentation of DNA-nuclear protein complexes in 1.9 M salt-neutral sucrose gradients (nucleoid sedimentation) was used to examine the effects of the DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) on mouse leukemia cell DNA. Mild detergent cell lysis and neutral pH make nucleoid sedimentation an extremely gentle, but sensitive, method to detect DNA scission. DNA breaks reduce the compaction of nucleoids and slow their sedimentation. Nucleoids from m-AMSA-treated cells sedimented as did those from untreated cells, indicating no detectable m-AMSA-dependent alterations in compaction despite an apparent underlying DNA break frequency of approximately 3 per 10(6) nucleotides, as measured by alkaline elution with proteinase. Mild proteinase digestion of cell lysates prior to nucleoid sedimentation unmasked some, but not all, of the underlying breaks. The frequency of DNA-protein cross-links in nucleoids from cells treated with m-AMSA was comparable to the single-strand break frequency produced by m-AMSA in whole cells. These results indicate that m-AMSA-induced DNA-protein cross-links conceal DNA breaks so as to prevent swiveling around the breaks within the nucleoids. This unique sort of DNA scission is consistent with the involvement of topoisomerases in the DNA breaks elicited by intercalators in mammalian cells.     

Nucleoid Condensation and Cell Division in Escherichia coli MX74T2 ts52 After Inhibition of Protein Synthesis

The reorganization of the bacterial nucleoid of an Escherichia coli mutant, MX74T2 ts52, was studied by electron microscopy after protein synthesis inhibition by using whole mounts of cell ghosts, ultrathin-sectioning, and freeze-etching. The bacterial nucleoid showed two morphological changes after chloramphenicol addition: deoxyribonucleic acid (DNA) localization and DNA condensation. DNA localization was observed 10 min after chloramphenicol addition; the DNA appeared as a compact, solid mass. DNA condensation was observed at 25 min; the nucleoid appeared as a cytoplasm-filled sphere, often opened at one end. Ribosomes were observed in the center. Giant nucleoids present in some mutant filaments showed fused, spherical nucleoids arranged linearly, suggesting that the tertiary structure of the nucleoid reflects the number of replicated genomes. Inhibitors which directly or indirectly blocked protein synthesis and caused DNA condensation were chloramphenicol, puromycin, amino acid starvation, rifampicin, or carbonyl cyanide m-chlorophenyl hydrazone. All inhibitors that caused cell division in the mutant also caused condensation, although some inhibitors caused condensation without cell division. Nucleoid condensation appears to be related to chromosome structure rather than to DNA segregation upon cell division.     

Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure

A strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas filamentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane-associated nucleoids were isolated from the wild-type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild-type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing influences on the organization of the bacterial nucleoid.     

Novel aspects of the structure of the Escherichia coli nucleoid investigated by a rapid sedimentation assay

The Escherichia coli nucleoid is maintained in its folded highly condensed state by constraints which involve RNA and protein. We have developed a rapid sedimentation assay to determine the state of folding of the membrane-free nucleoid. An approximate measure of the stability of the nucleoids under various conditions can then be estimated by measuring the temperature at which the nucleoids unfold. Using ethidium and gamma irradiation (which removes the negative supercoiling of the native nucleoid) as probes, it can be shown that there are two types of constraint involved in the condensation of the nucleoid. One of these constraints is destabilized by ethidium but stabilized by negative supercoiling; the second constraint is unaffected by both ethidium and negative supercoiling. Several models can be proposed: (i) a DNA . RNA duplex, (ii) a double-strand DNA (dsDNA) . RNA triplex, (iii) DNA-protein interactions, (iv) a topological knot with RNA, and (v) a DNA tetraplex. The topological knot model is not consistent with the data and many combinations of the others can be excluded. If RNA is involved in both constraints then RNA . DNA duplexes and dsDNA . RNA triplexes are involved in stabilizing the nucleoid.     

A simple autoradiographic method for investigating long range chromosome substructure: size and number of DNA molecules in isolated nucleoids of Escherichia coli.

A method is described for gently dissociating large DNA-protein complexes and for visualizing and quantitating the substructures by autoradiography. Using this technique, it is shown that nucleoids isolated from exponentially growing Escherichia coli (mean generation time = 35 min) contain on average 2.8 genome equivalents of DNA and that this nucleoid can be dissociated by deproteinization into two substructures having on average 1.4 genome equivalents. This result is correlated with previous sedimentation studies on the unfolded nucleoid DNA to explain prior inconsistencies. Scanning electron microscopy studies demonstrate that the shape and size of the isolated nucleoid is consistent with the proposed subunit structure of the in vivo nucleoid.