The effect of UV irradiation on the capacity of an Hfr recA strain of Escherichia coli to act as donor

Summary The ability of a recA Hfr strain of Escherichia coli to form colonies is extremely sensitive to inhibition by ultraviolet light (Fig. 2). Furthermore, in this strain the synthesis of DNA is stopped completely by a dose of 385 ergs/mm² of UV (Fig. 3). Nevertheless, the ability of this recA Hfr strain to act as a donor in sexual recombination was no more sensitive to UV than that of a wild type donor (Fig. 1). Furthermore, when irradiated and mated with a recA female, in which DNA synthesis was also inhibited by UV (Fig. 3), there was a net synthesis of DNA as measured by the incorporation of C¹⁴ thymidine (Fig. 4). By using nalidixic acid resistant recA donors and recipients in all combinations, irradiating with UV and treating with nalidixic acid during mating, it is shown that DNA was synthesized by the donor (Fig. 5). It is concluded that synthesis of DNA directed by the sex factor during mating in a recA donor is not as sensitive to inhibition by UV as normal DNA synthesis in a recA donor.     

Macromolecule synthesis leading to cell division in Tetrahymena pyriformis after replacement of required amino acids

Summary Tetrahymena pyriformis W were brought to a nonmultiplying state by removal of required amino acids from their growth medium. After amino-acid replacement, the incorporation rates of H³-uridine, H³-thymidine and H³-leucine were measured by the autoradiographic method. Following amino-acid replacement, the first response was detected in RNA synthesis, then protein synthesis, then DNA synthesis and, lastly, in cell division. Amino-acid deprived cells showed a 23% net increase in DNA content, a result supporting the view of others that protein synthesis is not necessary for the initiation of DNA synthesis but is necessary for the maintainance of DNA synthesis.     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Possible involvement of DNA-repair events in the transdifferentiation of mesophyll cells ofZinnia elegans into tracheary elements

In cultures of isolated mesophyll cells ofZinnia elegans, transdifferentiation into tracheary elements is induced by a combination of auxin and cytokinin and is blocked by inhibitors of DNA synthesis and poly (ADP-ribose) synthesis. During transdifferentiation, a very low level of synthesis of nuclear DNA was found in some cultured cells by microautoradiography after pulse-labeling with [³H]thymidine. Density profiles of nuclear DNA that had been double-labeledin vivo with bromodeoxyuridine (BrdU) and [³H]thymidine indicated that this DNA synthesis was repair-type synthesis. The sedimentation velocity of nucleoids increased during the culture of isolated mesophyll cells and the increase was dependent on phytohormones. This phenomenon may reflect the rejoining of DNA strand breaks after repair-type DNA synthesis during transdifferentiation. Treatment of cells with inhibitors of DNA synthesis or of poly(ADP-ribose) synthesis prevented the increase in the sedimentation velocity of nucleoids. The data suggest the involvement of DNA-repair events in the transdifferentiation of mesophyll cells into tracheary elements.     

DNA polymerase III-dependent repair synthesis in response to bleomycin in toluene-treated Escherichia coli

Summary Bleomycin (BLM) is an antitumor drug which interacts with and damages DNA. We have reported a repair response dependent on DNA polymerase I in toluene-treated Escherichia coli. We report here that DNA polymerase III can also catalyze a repair response in toluene-treated E. coli following exposure to BLM. Polymerase III-mediated synthesis differs because it is ATP-dependent, whereas polymerase I-mediated repair synthesis is not. Polymerase III repair synthesis is independent of replicative synthesis, as demonstrated in a polA ^-, dnaB _ts strain, or use of Novobiocin to inhibit replication, and replication persists in the presence of repair synthesis. It appears that ATP-dependent repair synthesis in response to BLM is also present in polA ⁺ strains. Repair synthesis does not require the uvrA gene product.This research was supported by Public Health Service grants GM-19122 and GM-24711 from the National Institute of General Medical Sciences, Robert A. Welch Foundation grant Q-543 and American Cancer Society BC-290     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

Restriction in vivo

Summary Degradation products of restricted T4 DNA induced filamentation, mutagenesis, and to a lesser extent, synthesis of recA protein in wild type cells but not in recA, lexA or recBC mutants of Escherichia coli. We conclude that the structural damage to the DNA caused by restriction cleavage and exonuclease V degradation can induce SOS functions. Degradation of restricted nonglucosylated T4 DNA by exonuclease V delayed cell division and induced filament formation and mutagenesis in lexA ⁺ but not in lexA ^- cells. Delay of cell division was also dependent upon recA and recBC funtions. Such degradation of DNA also dramatically increased mutagenesis in tif ^- Sfi^- cells at 42°C. The synthesis of recA protein continued in the restricting host after infection by the nonglucosylated T4 phage, but enhanced synthesis is not induced to the extent seen in SOS induced tif ^- cells grown at 42°. We also found that restriction of nonglucosylated T4 was alleviated in UV irradiated cells. The UV induced alleviation of rgl and r _K restriction depended upon post irradiation protein synthesis and was not observed in recA, lexA or recBC mutants.     

Properties of the nonlethal recombinational repair deficient mutants of bacteriophage T4

Summary The rate at which ³H thymidine is incorporated into DNA is increased in T4w-infected cells compared to wild-type when measured late in infection under conditions of low thymidine concentration. This increased DNA synthesis is sensitive to hydroxyurea but not to mitomycin C, and can be prevented by the addition of chloramphenicol early in infection. Also, DNA replicative intermediates isolated from T4w-infected cells late in infection sediment significantly faster than those isolated from wild-type-infected cells. In contrast, DNA replicative intermediates isolated from T4x-or T4y-infected cells sediment more slowly than those produced by wild-type T4. Cells coinfected with wild-type T4⁺ and T4x, y or w; or T4w and T4x or y, produce wild-type DNA replicative intermediates. Cells coinfected with T4x and T4y produce more slowly sedimenting DNA replicative intermediates. Cells coinfected with T4w and wild-type T4 show wild-type rates of DNA synthesis while cells coinfected with T4w and T4x or T4y show increased rates of DNA synthesis over that observed with wild-type alone.     

DNA synthesis inEscherichia coli B/r after UV-irradiation

When studying the kinetics of DNA synthesis, growth and cell division inEscherichia coli B/r after irradiation with different doses of UV-radiation (254 nm) we could demonstrate, by means of pulse incorporation of³H-thymidine, a lag in DNA synthesis after the irradiation. The relative rate of the restored DNA synthesis (related to the number of viable cells) was higher than in the non-irradiated culture. After 3 h the rate of DNA synthesis settled at a constant value, which was identical with the control rate up to the “critical dose” of 20 J/m². The irradiated cell population is heterogenous and contains basically two categories of cells — surviving and non-surviving. Cells of both types contribute to DNA synthesis restored after the lag period to a different extent. During the first hour after the irradiation even the nonviable portion of the population,i.e. cells that do not form colonies but are still penicillin-sensitive, is involved in the DNA synthesis.     

On the mechanism of conjugation in Escherichia coli K 12

Summary The phenomenon of conjugation consists of many stages. The most important are: the formation of contacts between mating cells, the transfer of DNA from the donor to the recipient, and the integration of the transfered DNA fragments into the chromosome of the recipient. Only after completion of all these stages are recombinants formed. With the aid of specific inhibitiors (nalidixic acid, FUDR), thymine starvation, and use of special thermosensitive mutants it is possible to study the role of DNA synthesis during every stage of conjugation. It was demonstrated that the genetic transfer is due to semiconservative DNA-replication in the donor cell. The fragments of DNA transfered are synthesized in the period of mating by a special replication system (F-replicon). In case of T _DNA ^S mutants unable to grow at 41°, the ability to synthesize DNA during conjugation is preserved.The inhibition of the DNA synthesis in the donor cell by poisons leads to complete inhibition of genetic transfer. The third stage — formation of recombinants requires DNA synthesis in the recipient cell and is inhibited by poisoning, thymine starvation or T _DNA ^S mutations in the recipient. In cases where recombination is not involved (i.e. sexduction) the inhibition of DNA synthesis in the recipient has no significant effect.     

An artifact in measurement of S phase initiation and its implication for the kinetics of S phase-specific enzyme activities

It is shown that the different onset of S phase as measured by autoradiography vs cumulative thymidine uptake is an artifact. We consequently propose that S phase-specific enzyme activities may accumulate a few hours prior to the actual initiation of DNA synthesis. A “pre-S” DNA synthesis that can be readily detected only by autoradiography has been proposed. Published data show that DNA synthesis in cultured animal cells is initiated approx. 2 h later when measured by cumulative incorporation of [³H]thymidine ([³H]TdR) as compared with autoradiography. We show here that the difference is in reality an artifact, owing to not taking into account both gradual, asynchronous entry of cells into S phase, as well as time-dependent accumulation of radioactivity into each cell after it has entered S phase. Combination of these two factors leads to the conclusion that [³H]TdR should be incorporated approximately as the square of time following entry of the first cell into S. Taking this into account, the two methods then are in agreement, as predicted. This argument also applies to the enzyme activities shown to increase with DNA synthesis in synchronized cultures. Such an enzyme accumulation really could begin some time earlier than indicated by conventional plots of cumulative enzyme activity vs time and may, in fact, precede the onset of S by a few hours.     

The occurrence and role of DNA synthesis during meiosis in wheat and rye

The incorporation of ³H-thymidine into the DNA of rye meiocytes at zygotene, pachytene-diplotene and metaphase I to telophase II stages has been studied. Low levels of ³H were found in highly purified DNA from meiocytes at all these stages, though there was more in the DNA from pachytene-diplotene meiocytes, and it is highly likely that the zygotene groups of anthers contained a proportion at pachytene. The buoyant density distributions of the labelled DNA from zygotene and pachytene-diplotene cells were indistinguishable, in contrast to the situation in Lilium, the only other example studied so far.The DNA synthesis inhibitor 2′-deoxyadenosine halted meiotic development of anthers in culture only at late zygotene and pachytene. It did not inhibit development at early zygotene, prevent chromosome pairing as judged by light microscopy or cause extensive chromosome fragmentation during zygotene as in Lilium. These results indicate that extensive synthesis of DNA does not occur at zygotene in cereals and does not suggest that zygotene DNA synthesis is a prerequisite for chromosome pairing as in Lilium.     

DNA replication in homogenates of Physarum polycephalum

Homogenates of Physarum polycephalum incorporate [³H] dATP into nuclear DNA at an initial rate of approximately 15% of the in vivo rate. To attain this level of synthesis, cultures are homogenized in a medium containing Mg⁺⁺, EGTA, glucose and spermine. Incorporation is strongly stimulated by the addition of ATP and all four deoxyribonucleoside triphosphates to homogenates prior to incubation. Various inorganic cations other than Mg⁺⁺ either do not affect synthesis or are inhibitory. Incorporation is inhibited by a nonionic detergent, Triton X-100. DNA synthesis in this cell-free nuclear system is similar in several respects to that which occurs in vivo: (1) The rate of DNA synthesis in the intact organism at a given time in the mitotic cycle is reflected by the level of synthesis in homogenates prepared from cultures at that time of the cycle; (2) DNA strands labeled in vitro exhibit alkaline sucrose density gradient sedimentation properties similar to those of daughterstrand DNA pulse-labeled in vivo; and (3) Homogenates of cultures which were pre-treated with cycloheximide incorporate [³H]dATP at about 60% of the level observed in homogenates of untreated controls.     

The heterodimeric primase from the euryarchaeon Pyrococcus abyssi: a multifunctional enzyme for initiation and repair?

We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase α–primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg²⁺ and Mn²⁺. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.     

G2 arrest and differentiation in the petal of Tradescantia clone 4430

The floral organs of Tradescantia clone 4430 were used to investigate, in terms of cell cycle parameters, cellular behaviour during the maturation of a terminally differentiating system. Petals were sampled at different stages of development for (a) cell number; (b) nuclear DNA content by cytophotometry; (c) [³H]thymidine incorporation into nuclei by autoradiography; and (d) pigment production by spectrophotometry. DNA synthesis was confirmed by measurement of [³H]thymidine incorporation into TCA-insoluble material and changes in DNA content by colorimetric estimation of DNA extracts by diphenylamine. The development of the petal involved four sequential steps. First, there was an increase in cell number, an event characterized by mitoses, DNA synthesis, a few cells in G2 and a predominance in G1. Second, there was a cessation of cell division and DNA synthesis when all the cells accumulated in G1. Third, there was a shift of a large proportion of the total cell population from G1 to the G2 stage of the cell cycle and finally, there was pigment production. In addition, cytophotometric analysis of individual tissues in the mature petal revealed tissue specific differences in the proportion of cells in G2.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.     

Lambda red-mediated synthesis of plasmid linear multimers in Escherichia coli K12

Summary Expression of the red ⁺ and gam ⁺ genes of bacteriophage in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red ⁺ or gam ⁺ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam ⁺ or red ⁺ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red ⁺ products, protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD^– Exol^– cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3 singles-tranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.Abbreviations PLM plasmid linear multimers - BUDR 5-bromo-2-deoxyuridine - bp base pair     

Metal ion induced unscheduled DNA synthesis in Petunia pollen

Summary Al³⁺, Fe³⁺, V²⁺ or Be²⁺ when added to shaken suspensions of Petunia hybrida pollen in 10% sucrose —0.01% H₃BO₃ induce a strong unscheduled DNA synthesis as measured by incorporation of ³H-thymidine into pollen DNA. The metal ions (added in most cases as the chloride) gave maximum effect at approximately 2 mM. Weaker reactions are given by Ca²⁺, Zn²⁺, Mn²⁺, Cd²⁺ and Cr³⁺ in decreasing order of effectiveness, while twelve other metal ions were shown to be ineffective or to give very low reaction. The unscheduled DNA synthesis induced by Al³⁺ was not altered by hydroxyurea, nicotinamide, caffeine or cycloheximide. It was markedly affected by the pH of the medium, the optimum pH being 5.0, where there could be a tendency for some base-binding of the metal (in contrast to phosphate binding) at the high Al³⁺ to DNA mole ratio used. It was considered that the DNA synthesis induced by the metal ions represents a repair synthesis. A DNA polymerase activity was detected in pollen extracts. It showed a preference for Mn²⁺ over Mg²⁺ and was estimated to have more than enough activity to account for the unscheduled DNA synthesis in pollen given by the most effective inducer, Al³⁺.     

Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N-dG adducts by human DNA polymerases η and κ

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase η (Polη) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)-trans-anti-BPDE-N²-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)-trans-anti-BPDE-N²-dG adduct than opposite the (−)-trans-anti-BPDE-N²-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N²-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts.     

Qualitative and Quantitative Studies on DNA and RNA Synthesis in Loxodes striatus Nuclei*

SYNOPSIS. In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei are differentiated morphologically into a clearly recognizable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a ³H-labeled precursor ([³H]thymidine for DNA and [³H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [³H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [³H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macro-nucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components of the nuclear complex were investigated. (F) Contrary to the previously reported information our results suggest that DNA synthesis can occur in adult macronuclei. The possible explanation of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.