Genomic landscape of single-stranded DNA gapped intermediates in Escherichia coli

Single-stranded (ss) gapped regions in bacterial genomes (gDNA) are formed on W- and C-strands during replication, repair, and recombination. Using non-denaturing bisulfite treatment to convert C to U on ssDNA, combined with deep sequencing, we have mapped gDNA gap locations, sizes, and distributions in Escherichia coli for cells grown in mid-log phase in the presence and absence of UV irradiation, and in stationary phase cells. The fraction of ssDNA on gDNA is similar for W- and C-strands, ∼1.3% for log phase cells, ∼4.8% for irradiated log phase cells, and ∼8.5% for stationary phase cells. After UV irradiation, gaps increased in numbers and average lengths. A monotonic reduction in ssDNA occurred symmetrically between the DNA replication origin of (OriC) and terminus (Ter) for log phase cells with and without UV, a hallmark feature of DNA replication. Stationary phase cells showed no OriC → Ter ssDNA gradient. We have identified a spatially diverse gapped DNA landscape containing thousands of highly enriched ‘hot’ ssDNA regions along with smaller numbers of ‘cold’ regions. This analysis can be used for a wide variety of conditions to map ssDNA gaps generated when DNA metabolic pathways have been altered, and to identify proteins bound in the gaps.     

Changes in bacterial cells induced by high pressure at subzero temperature

The aim of this study was to examine the effect of pressure treatment at 193 MPa and −20 °C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.     

The Dampening of DNA Replication Cycles after X-Irradiation or Ultraviolet Light Irradiation

Escherichia coli strains 15T^- (555-7) and B/r were grown in the presence of thymine-¹⁴C to label all DNA. The ability of these parental DNA's to undergo cycles of replication subsequent to cellular irradiation with either X-ray or ultraviolet light (UV) was followed with density labels. Exposed cells were shifted into the density medium at times which were approximately multiples of normal rounds of DNA replication. A portion of the parental DNA, replicated semiconservatively once during an initial cycle following UV or X-irradiation in E. coli, failed to replicate again within the time studied. The time course of semiconservative parental DNA replication is altered.     

Role of Lamin B1 in Chromatin Instability

Nuclear lamins play important roles in the organization and structure of the nucleus; however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. The S phase delay appears to be due to the stalling and collapse of replication forks. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions.     

Rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after cessation of dihydrofolate reductase activity in stationary versus log phase L1210 leukemia cells.

Previous studies from this laboratory established that the rapid but partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure of L1210 leukemia cells to antifolates cannot be due to direct feedback inhibition of thymidylate synthase by dihydrofolate or any other endogenous folylpolyglutamates when dihydrofolate reductase activity is abolished by antifolates. Rather, the data suggested this preservation of tetrahydrofolate cofactor pools is likely due to a fraction of cellular folates unavailable for oxidation to dihydrofolate. This paper explores the role of cell cycle phase in L1210 leukemia cells in logarithmic versus stationary phase growth as a factor in the rate and extent of tetrahydrofolate cofactor interconversion to dihydrofolate after exposure of cells to the dihydrofolate reductase inhibitor trimetrexate. The S phase fraction was reduced by inoculating L1210 leukemia cells at high density to achieve a stationary state. Flow cytometric analysis of DNA content indicated that log phase cultures were 53.0% S phase; this decreased to 42.1% at 24 h and 24.1% at 48 h in stationary phase cultures. 5-Bromo-2'-deoxyuridine incorporation into DNA decreased 80 and 96%, while [3H]dUrd incorporation into DNA declined 70 and 95% for stationary cultures at 24 and 48 h, respectively, as compared with the log phase rates. Log phase cells interconverted 28.0% of the total pool of radiolabeled folates to dihydrofolate with a half-time of approximately 30 s. Stationary cells at 24 h interconverted 20.4% of the total folate pool with a t1/2 of approximately 3 min, and at 48 h, net interconversion to dihydrofolate decreased further to 12.1% with a t1/2 of approximately 6 min. The decrease in the extent of tetrahydrofolate cofactor interconversion to dihydrofolate in stationary phase cells was directly proportional to the decrease in the S phase fraction determined by total DNA content. This suggests that tetrahydrofolate cofactor depletion occurs only in S phase cells. The much larger drop in [3H]dUrd and 5-bromo-2'-deoxyuridine incorporation into DNA in comparison with the decline in the S phase fraction measured by DNA content along with the reduced rate of tetrahydrofolate cofactor interconversion to dihydrofolate indicates that the rate of DNA synthesis is decreased in S phase cells in stationary cultures. Network thermodynamic simulations suggest that a reduction in the number of S phase cells and their thymidylate synthase catalytic activity would account for the observed decrease in the rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after trimetrexate in stationary phase cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Replication of DNA: III. Changes in the Number of Strands in E. coli DNA during Its Replication Cycle

DNA has been isolated from synchronized cultures of E. coli 15_T- at various times. At first the DNA was four-stranded (and indistinguishable in all respects from log phase E. coli DNA), but at the start of DNA synthesis the DNA was found to have halved its molecular weight and to have become two-stranded. This sample had all the properties of undenatured, double-helical DNA, and behaved in all respects like DNA from non-proliferating sources. The replication cycle of the DNA molecule has thus been shown to consist of an alternation between the four- and two-stranded forms, the latter being the conserved unit. The evidence provided by the three papers of this series with respect to DNA and chromosomal structure and replication is discussed and summarized.     

Adsorption of coliphages T1 and T7 to host and non-host microbes and to clay minerals

The clay minerals, kaolinite (K) and montmorillonite (M), suspended in either distilled water (DW) or a minimal medium (M-9), were better adsorbents for coliphages T1 or T7 than were bacteria (including early log, late log, or stationary phase cultures of the hosts), actinomycetes, and yeasts. Except for the host bacteria, the microbial cells (regardless of their type, phase of growth, viability, weight or number of cells, and volume of the suspension medium) adsorbed few or no coliphages. Although early log phase cultures (3 h) ofEscherichia coli B, suspended in DW, adsorbed an appreciable amount of T1 (94%), washing the cells with DW reduced the amount of T1 adsorbed (48%); 3-h cultures ofE. coli B/1,5, suspended in DW, adsorbed 15% of a T7 inoculum, and washing the cells with DW reduced the amount of T7 adsorbed to 1%. There was appreciable adsorption (35 to 97%) of both coliphages (with the exception of T1 on K) to 1 mg K or M suspended in either DW or M-9. These results suggest that clays are more important than microbes as adsorbents of viruses in environments of low ionic strength and that microbes do not inactivate coliphages T1 and T7.     

The regulation of DNA replication during senescence and rejuvenation of Dunaliella tertiolecta by factors other than available metabolic energy

The unicellular green alga, Dunaliella tertiolecta undergoes a 4-fold reduction in DNA while progressing from early to late log phase of culture. During the period when the reduction in DNA occurs, the cells continue to divide at the maximal rate. Pulse labelling indicates little incorporation of [³H]thymidine into DNA during late log phase. Stationary phase cultures diluted with fresh medium undergo a lag period during which there is a 4-fold increase in DNA and a rapid incorporation of [³H]thymidine into DNA into DNA before division. The evidence indicates that the differences in the levels of DNA are not attributable to tetraploidy, multinucleated cells or a high level of redundancy of G-C-rich satellite DNA in early log phase cells. During stationary phase there is an increase in cellular starch and a decrease in free nucleotides. Electron microscopy reveals that stationary phase cells are distorted by many granules proven to be starch by their susceptibility to amylase treatment. The production of starch by stationary phase cells indicates that DNA replication does not cease because of a deficit of metabolic energy but because of some direct function of culture density.     

Arrest of cell division blocks the utilization of polyamines in synchronized cultures of photoautotrophically grown Euglena

A trimodal change in the cellular levels of three major polyamines: spermidine, N,N′-bis(3-aminopropyl)-1, 3-propanediamine (BAP) and 3,3′-diaminodipropylamine (DAD) was observed during two successive cell cycles in synchronously dividing cultures of the algal flagellate, Euglena gracilis Z photoautotrophically grown in a 24-h light-dark cycle. The intracellular levels of these three polyamines decreased as cells divided and then were enhanced as cells exited the G₁ phase and proceeded through the S and G₂ phases. Spermidine, BAP and DAD concentrations increased about 2.5-fold during the S phase. Putrescine and 1,3-diaminopropane levels did not vary significantly. One peak of polyamine synthesis occurred in the G₁ phase prior to DNA synthesis, followed by a second more important peak during the S-G₂ phases before cell division; both peaks were observed during the light period. A third minor peak was observed during the pre-G₁ (or G₀) phase in the dark period after mitosis had been completed. In contrast, when the cells attained the “stationary” phase of growth, there was no significant increase in the content of polyamines during the light period although spermidine and BAP increased slightly twice during the dark period (putrescine and 1,3-diaminopropane and DAD levels remained almost constant). To ascertain whether the synthesis of polyamines was merely a direct effect of the photoperiod, parallel experiments with synchronous cultures were carried out in the presence and absence of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, a photosynthetic inhibitor. Although a slight decrease in the concentration of polyamines was observed, the three maxima of polyamines synthesis were observed as in normal cultures. These results clearly suggest that polyamine biosynthesis is closely related to DNA replication and cell division in Euglena cells.     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Uptake of bacterial DNA by Chlamydomonas reinhardi

Escherichia coli [³H]DNA supplied to vegetative cultures of wild-type (mt+) and CW15 (mt+; mutant lacking the cell wall) Chlamydomonas reinhardi could bind to the cell wall of the wild-type and to the cell membrane of CW15 mutant cells. The extent of this binding decreased with time and was to a large degree (over 90%) DNA-ase-sensitive. Nevertheless, about 0.01% of the bacterial DNA remained irreversibly associated with the cells when they reached stationary phase. The irreversible binding of the donor bacterial DNA to Chlamydomonas cells could be increased by treatment of the cultures with polycations such as DEAE-dextran, poly-L-lysine and poly-L-ornithine. Although the CW15 cells rapidly degraded bacterial DNA in the culture medium wild-type cells showed only a small effect on the molecular weight of the donor DNA.The acid-insoluble radioactivity irreversibly bound to WT (+) cells consisted mainly of oligonucleotides with a small proportion present as less depolymerized donor DNA. No radioactivity, however, was found to be associated with the recipient high molecular weight Chlamydomonas DNA.No labeled donor DNA could be recognized in the cells given bacterial [³H]DNA in early stationary phase. Instead, radioactivity found in Chlamydomonas DNA corresponded to reutilization of [³H]thymine derivatives released as a result of [³H] DNA degradation. No evidence for the integration of detectable amounts of donor DNA sequences into the host cell DNA was obtained.     

Control of Saccharomyces cerevisiae 2microN DNA replication by cell division cycle genes that control nuclear DNA replication.

The replication of the 2 μm DNA of Saccharomyces cerevisiae has been examined in cell division cycle (cdc) mutants. The 2 μm DNA does not replicate at the restrictive temperature in cells bearing the cdc28, cdc4, and cdc7 mutations which prevent passage of cells from the G1 phase into S phase. Plasmid replication also is prevented in a mating-type cells by α factor, a mating hormone which prevents cells from completing an event early in G1 phase. The 2 μm DNA ceases replication at 36 °C in a mutant harboring the cdc8 mutation, a defect in the elongation reactions of nuclear DNA replication. Plasmid replication continues at the restrictive temperature for approximately one generation in a cdc13 mutant defective in nuclear division. These results show that 2 μm DNA replication is controlled by the same genes that control the initiation and completion of nuclear DNA replication.     

Cell Reproduction and Morphological Changes in Mycoplasma capricolum

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.Mycoplasmas are parasitic bacteria that have extremely low G+C contents and small genomes (9). Their morphology is irregular because of the lack of a peptidoglycan layer.In Escherichia coli, initiation of chromosomal DNA replication occurs once during the cell’s replicative cycle, and the nucleoids partition before cell division (13). The chromosomal replication of E. coli initiates in a small region and proceeds in both directions. It is mainly controlled by the timing and frequency of initiation, while the velocity of replication is constant.In mycoplasmas, chromosome replication also starts at a fixed site, followed by bidirectional progression (19–21, 25, 40). As in many eubacteria (36), the dnaA gene is expressed and plays important roles in the initiation of replication (35). These observations suggest that the outline of chromosome replication of mycoplasmas is similar to that of E. coli. However, the process of mycoplasma cell reproduction has not been clarified. Moreover, the cell division cycle of E. coli cannot be simply applied to mycoplasmas because of their irregular cell morphology (4). A model has been suggested for the cell cycle of Mycoplasma mycoides (6, 30, 31), which is closely related to Mycoplasma capricolum (39). According to this model, an elementary rounded body grows into a filamentous form and then new elementary rounded bodies are developed within this filament and released, but this model has not been adequately substantiated.In this study, we analyzed the process of DNA replication, cell morphology, and viability under various conditions of M. capricolum and proposed a model of cellular reproduction for this bacterium.     

Two-dimensional DNA electrophoresis applied to the study of DNA methylation and the analysis of genome size in Myxococcus xanthus

Methylation changes in the DNA of Myxococcus xanthus were studied using a twodimensional DNA electrophoresis technique in which one-dimensional polyacrylamide separations of HpaII digests of DNA extracted from different stages of development were re-digested in situ with MspI and then run in a second dimension. Specific methylation events were seen to be associated with the slowing down of cell growth as vegetative cells entered stationary phase, and also as cells on starvation agar progressed through developmental stages. Two-dimensional agarose electrophoresis was employed to obtain an unambiguous estimate of the genome size of this organism, approximately 5690 × 10³ base-pairs (±9%). Using the same method, the Escherichia coli genome was measured to be 3520 × 10³ base-pairs (±7%).     

Yeast plasmids have fewer superhelical turns than predicted by the nucleosome repeat of yeast DNA

The superhelical density of three Saccharomyces cerevisiae plasmids was determined with respect to a defined reference state during vegetative growth and stationary phase. The levels of supercoiling determined were approximately 20% lower than predicted by comparisons with SV40 DNA and reconstituted minichromosomes using histones from higher eukaryotes. In two different plasmids with the ARS1 origin of replication, the level of supercoiling changed substantially as the host cells entered stationary phase. Supercoiling of the endogenous 2-microns plasmid during vegetative growth was lower than in the ARS1-containing plasmids but did not change significantly upon entry of the cells into stationary phase.     

The recovery of normal DNA replication kinetics in ultraviolet-irradiated Chinese hamster cells

The kinetics of DNA replication were analyzed in the second S phase following UV irradiation of Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by treating cells selected in mitosis with hydroxyurea for 9 h. Following UV irradiation, the cells were allowed to progress until the next mitosis; at which time they were resynchronized at the beginning of the second S phase by the same procedure. The kinetics of DNA replication were determined by measuring the proportion of DNA which achieved hybrid buoyant density on CsCl density gradients as a function of the time of incubation in the presence of 5-bromodeoxyuridine.The results of these experiments showed that even though the rate of DNA replication is substantially depressed during the first S phase following UV irradiation with a fluence of 5 J/m², the rate has recovered to the extent that it is indistinguishable from the unirradiated control by the time the cells have entered their second S phase. It was concluded from these observations that the lesions in DNA which caused the rate of DNA replication to be initially depressed during the first S phase have been either removed or modified such that they no longer are able to cause a reduction in the rate of DNA replication in the second S phase following UV irradiation.     

The dependency of nuclear division on volume in the dimorphic yeast Candida albicans

When stationary phase cells of the dimorphic yeast Candida albicans are induced to synchronously form mycelia, over 90% of the cells undergo nuclear division. However, when stationary phase cells are induced to synchronously form buds, less than half undergo nuclear division even though all form buds. The majority of cells which do not undergo nuclear division form buds with volumes below a threshold value and the majority of cells which do undergo nuclear division form buds with volumes above this threshold value. In this report, we have investigated the possibilities that cells which form small buds do not attain a particular mass threshold. Cell cultures were examined for DNA replication, dry weight, and protein content during synchronous bud and during synchronous mycelium formation. Evidence is presented which indicates that the lack of nuclear division in over half of a budding population is due to low daughter cell volumes or to low surface areas, and not to their failure to attain a mass threshold or to replicate their DNA. The dependency of nuclear division on daughter cell volume is discussed.     

Fundamental structural units of the Escherichia coli nucleoid revealed by atomic force microscopy

A small container of several to a few hundred µm³ (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of ~80 nm was found. In addition to this ‘80 nm fiber’, a thinner ‘40 nm fiber’ and a higher order ‘loop’ structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a ‘coral reef structure’ that also possessed the 80 nm fiber units, and, finally, into a ‘tightly compacted nucleoid’ that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid.     

Effects of cis-platinum(II) diamminedichloride on survival and the rate of DNA synthesis in synchronously growing HeLa cells in the absence and presence of caffeine

Synchronously growing HeLa cells demonstrated a different profile of DNA synthesis to that observed for Chinese hamster V79-379A cells after treatment with cis-Platinum(II) diamminedichloride (cis-Pt(II)) in the G₁ phase of the cell cycle. The progression of G₁ phase treated cells into the DNA synthetic phase was not affected. The peak rate of DNA synthesis in the first cycle was decreased in a dose dependent manner. However, no displacement in the time of appearance of this peak rate of DNA synthesis was observed in the first cycle as had been observed in Chinese hamster V79-379A cells. The timing of mitosis after the first cycle was delayed in a dose dependent manner and resulted in a concomitant delay in the appearance of the peak rate of DNA synthesis in the second cycle. The peak rate of DNA synthesis in the second cycle was reduced in a dose dependent manner. The ability of cells to divide after the first cycle was not related to their eventual ability to survive. Incubation of HeLa cells with caffeine after treatment with cis-Pt(II) did not increase the toxicity of cis-Pt(II). This was consistent with the lack of effect of caffeine posttreatment on the rate of DNA synthesis in cis-Pt(II) treated synchronously growing HeLa cells. HeLa cells did not show the characteristics of caffeine sensitive replication repair, nor did they show evidence for the presence of an inducible repair system. The rate of DNA synthesis, cell number and survival data were discussed in relation to a mechanism of cell death proposed for Chinese hamster cells.