Growth-medium-dependent repair of DNA single-strand and double-strand breaks in X-irradiated Escherichia coli

The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N. J. Sargentini, W. P. Diver, and K. C. Smith, Radiat. Res. 93, 364-380, 1983). In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair. While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair. This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis. Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process. Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast. Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA. With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.     

The origin of nascent single-stranded DNA extracted from mammalian cells.

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.     

ACCUMULATION OF REPLICATIVE INTERMEDIATES OF MITOCHONDRIAL DNA IN TETRAHYMENA PYRIFORMIS GROWN IN ETHIDIUM BROMIDE

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10–15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.     

Cellular Origin of a Mouse Leukemia Viral Ribonucleic Acid

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.     

Requirements for extraction of polyribosomes from plant callus cultures

Zone sedimentation through sucrose gradients was used for preparing Rhizobium bacteroids from lupin nodules and for separating them into slowly and rapidly sedimenting fractions.     

Sedimentation velocity of DNA in isokinetic sucrose gradients: calibration against molecular weight using fragments of defined length.

The relationship between sedimentation coefficient and molecular weight for DNA sedimenting in preformed alkaline and neutral sucrose gradients was determined using absolute molecular weight standards (restriction fragments of plasmid pBR322 and phage lambda DNA). The range of calibration for alkaline gradients was extended to small DNA fragments (652 base-pairs) for the first time. The exponent b in the equation S20 degrees, w = aMb was found to be 0.380 in neutral gradients and 0.410 in alkali. The latter value differs significantly from previous estimates. The gradients were isokinetic, and the distance sedimented was shown to be directly proportional to the sedimentation coefficient at all times.     

Inhibition of initiation of HeLa cell replicons by methyl methanesulfonate.

Methyl methanesulfonate (MMS) in the culture medium inhibits the rate of DNA synthesis in HeLa cells in a dose-dependent manner. By using short (5 min) incubations with [3H]thymidine and analyzing the newly made DNA by velocity sedimentation on alkaline sucrose gradients, we found that the first affect of MMS on DNA replication, at 0.5 h after treatment, was inhibition of initiation of replicons. Recovery from this effect seemed to have begun by 2 3/4 h after treatment. The second effect of MMS, which was evident at 2 h after treatment, was to slow or block chain elongation.     

DNA replication in methotrexate-treated human lymphoblasts.

The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5-bromodeoxyuridine-substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2-min or 10-min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate-treated cultures were pulse-labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk-size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation.     

Size and Composition of Marek''s Disease Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified nucleocapsids of Marek's disease herpesvirus (MDV) was cosedimented with T4 and with herpes simplex virus (HSV) DNA in neutral sucrose density gradients and with T4 DNA in alkaline sucrose density gradients. These experiments indicated that the intact MDV DNA had a sedimentation constant of 56S corresponding to a molecular weight of 1.2 x 10(8) daltons. In the alkaline gradients, the largest and most prominent band contains a DNA sedimenting at 70S corresponding to 6.0 x 10(7) daltons in molecular weight. The DNA is therefore double-stranded and not cross-linked. Isopycnic sedimentation of the MDV DNA molecules with SPO1, Micrococcus lysodeikticus, and HSV DNA gave a density of 1.705 g/cm(3) corresponding to 46 guanine plus cytosine moles per cent. Lastly, in hybridization tests the DNA hybridized with RNA of infected cells but not with that of uninfected cells supporting the conclusion that it is viral.     

Characterization of large mammalian DNA species sedimented in a reorienting zonal rotor

The characteristics of a Beckman-designed slow acceleration unit for the reorientation of alkaline sucrose gradients in a Ti-15 zonal rotor are described. The large DNA species (> 250S) obtained from cultured rat brain tumor cells with this system sediment linearly with time, have virtually no [³H]leucinelabeled or covalently bonded [³H]choline-labeled material sedimenting with them, sediment independently of smaller single-stranded DNA molecules (? 165S) and are 60–80% degraded by the single-strand-specific S1 nuclease. Therefore, it is postulated that these species are collapsed, partially denatured DNA molecules or a collapsed form of single-stranded DNA. When cells were labeled with [¹⁴C]TdR, then frozen and stored at ? 79°C, this system could detect radiation-induced DNA damage from decay of the incorporated label at accumulated doses as small as 18–126 rads.     

Influence of Thymine Starvation on the Integrity of Episomal and Chromosomal Deoxyribonucleic Acids in Escherichia coli CR34 (λ ind−)

The effect of thymine deprivation on the integrity of phage lambda, sex factor, and chromosomal deoxyribonucleic acid (DNA) in Escherichia coli CR34 (lambda ind(-)) was examined by sedimenting cell lysates through alkaline sucrose gradients. Both sex factor and chromosomal DNAs showed evidence of being degraded during the starvation period. In contrast, no loss of closed circular lambda DNA was observed.     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

Studies on the stability of chromatin from hen oviduct

The stability of sheared chromatin from the magnum portion of hen oviduct has been examined during storage at 0 °C using several parameters. Chemical composition and ultraviolet spectra were unchanged after 1 month while changes in melting profiles were seen after 2 weeks. Gel electrophoretic patterns of histones indicated slight proteolysis after 4 weeks with no apparent degradation occurring at earlier times. Acidic protein gel patterns remained unchanged during the period of study. Rapid changes (within 1 to 3 days) were seen in chromatin size distributions as measured in sucrose gradients and in chromatin template activity. The mean chromatin size decreased with storage, an increasing percentage of the total population sedimenting as a 17S component. This change in chromatin size was accompanied by an increase in the amount of free protein floating on the top of gradients.Several experiments were conducted to investigate the nature of the chromatin changes occurring at early storage times. Changes in chromatin size and template activity could be attributed to neither proteolysis nor DNase activity. Diisopropylfluorophosphate, a known nuclear protease inhibitor, did not inhibit the rate of change in chromatin size with storage. Furthermore, no proteolysis was detectable by gel electrophoresis during storage times at which significant changes in chromatin size and template activity had occurred. When the size of chromatin DNA was examined, no changes were seen with storage nor were there differences in the size of DNA from fast and slow sedimenting chromatin fractions.When template activities of fast and slow sedimenting chromatin fractions were compared, the slow sedimenting fractions were found to be the most template active. The template activity of a slow sedimenting, high template activity fraction was examined with storage and was found to increase with time.     

Strandedness and Complementarity of DNA from Long-Term RNA-Dependent DNA Polymerase Reactions of Soehner-Dmochowski Murine Sarcoma Virus

The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.     

Estimation of DNA fork displacement rates from isolated mammalian chromosomes

Chromosomes were isolated from Chinese hamster ovary cells that had been pulse-labeled with ³H-thymidine and bromodeoxyuridine and allowed to proceed to metaphase in the presence of colcemid. The chromosomes were then irradiated with 313 nm light and the length of the pulse-labeled DNA segments was estimated by the distribution of ³H-DNA in alkaline sucrose gradients. The average rate of fork displacement, calculated from this measurement, was 0.9 m/min, which was in excellent agreement with measurements from interphase cells. By the same method, hydroxyurea was shown to retard DNA chain growth by 50–60% in both isolated chromosomes and interphase cells.     

Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients

Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 x 10(6) daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 x 10(6) to 39 x 10(6) daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with (3)H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments.     

Synchronization of replicons in Ehrlich ascites cells

Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G1 cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 microM) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G1 cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles highly synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.     

Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (-) strand.

Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.     

Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

Rate of DNA chain elongation in ultraviolet light-irradiated mammalian cells as estimated by a bromodeoxyuridine photolysis method.

Using a new method, we show that, when the DNA chain elongation rate is measured for short time periods, ultraviolet light does not decrease this rate. The method is based on the photolysis of bromouracil-containing DNA by 313 nm light and alkaline sucrose gradients.