Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation <Emphasis Type="Italic">nrdA101</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> suggest an effect on chromosome segregation

NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA _ts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.     

A mutant ofLactobacillus acidophilus with temperature-sensitive regulation of DNA synthesis

Among other temperature-sensitive mutants ofLactobacillus acidophilus the mutant “ts 9” with temperature-sensitive initiation of DNA synthesis was isolated. In this mutant, the course of DNA synthesis under non-permissive conditions proceeds in two phases. During the first 90–120 min, a slight increase (20–50%) of DNA content takes place. Then during further incubation at 40°C, the capacity for initiation of further DNA synthesis increases and a second round of DNA synthesis starts after 3–4h of incubation. The initiation of DNA synthesis is prevented by chloramphenicol and the preceding lag is temperature-dependent. It is concluded that an accumulation of an initiation factor is required for the onset of a new cycle of DNA synthesis and that in thets 9 mutant this accumulation is inhibited at non-permissive temperature.     

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants ofUstilago maydis

Summary The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungusUstilago maydis after incubation at the restrictive temperature (32° C) for eight hours. Mutantsts-220,ts-207,ts-432 andts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutantsts-20,tsd 1-1,ts-84 andpol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutantpol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutantts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32°C.tsd 1-1 andts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis with correlates to increasing UV sensitivity of these strains on incubation at 32° C. Apol 1-1ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.     

Temperature sensitive mutations affecting RNA synthesis in Escherichia coli

Summary A streptomycin method has been used for the isolation of mutants with RNA synthesis inhibited at elevated temperature. The method is based on the observation that streptomycin kills bacteria with normal RNA synthesis and does not affect the cells with inhibited synthesis of RNA. This selection method increases the yield of temperature sensitive mutants by a factor 10–20, the amount of mutants with disturbed RNA synthesis is increased 3–5 fold as compared with the method of replicas.Several types of mutants were found among the temperature sensitive strains: those possessing temperature sensitivity of one, two or three types of cellular macromolecules DNA, RNA and protein. The screening among the mutants with affected RNA synthesis revealed a strain ts-19 showing low RNA polymerase activity in cell extracts and partially purified RNA polymerase preparations. The presented evidence suggests that ts-19 mutation affects the structural gene of one of the RNA polymerase subunits.The mapping of the corresponding locus indicated that it was located between the str and thy loci in E. coli K 12 chromosome at a distance of about 20 recombination units from the first locus.     

Mu DNA replication in vitro: Criteria for initiation

Summary An in vitro system for investigating Mu replication and transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates.Two conditions reported to specifically decrease Mu DNA replication in vivo (Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.     

Phenotypic Characterization of Adenovirus Type 12 Temperature-Sensitive Mutants in Productive Infection and Transformation

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxyl-amine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for “initiation” of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutated in H12ts505 is required to maintain at least some aspects of the transformed state.     

Functional analysis of the dna (Ts) mutants of Bacillus subtilis: plasmid pUB110 replication as a model system

Summary We determined the effect of various Bacillus subtilis dna(Ts) mutations on pUB110 and chromosomal replication. Leading strand DNA synthesis of pUB110, starting by a nick at the plasmid replication origin (oriU), is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in thermosensitive mutants dnaD, dnaF, dnaH and dnaN known to cause thermosensitivity of the various subunits of DNA polymerase III. When the lagging strand origin (oriL) is exposed, the DnaG protein (DNA primase) alone, or in association with unknown protein(s) binds asymmetrically to oriL to form a primer that is also extended by DNA polymerase III. In oriL ^- plasmids like pBT32, leading and lagging strand DNA syntheses are decoupled from each other. The DnaB protein, that is not required for pUB110 replication, may be associated with priming at a second unidentified lagging strand origin on pBT32. At non-permissive temperature, the dnaC30 and dnaI2 mutations affect both pUB110 and chromosomal DNA synthesis.     

Prophage induction in thermosensitive DNA mutants of Bacillus subtilis

Summary Incubation of thermosensitive dna mutants of Bacillus subtilis at the non-permissive temperature leads in some instances to induction of defective prophage PBSX and cell lysis. A clear distinction can be made between mutants affected in DNA replication at the growing point (extension mutants) and those unable to initiate new rounds of replication (initiation mutants). The former promote PBSX induction to a variable and mutation-specific extent, whereas the latter do not exhibit any signs of induction. Analysis of mutants carrying two dna mutations suggests that products of some dna genes involved in initiation and in extension are not essential for induction but can substantially amplify its extent. However, mitomycin C treatment of dna mutants which have completed their residual DNA synthesis leads to a PBSX induction essentially identical to that obtained by mitomycin C treatment of the wild-type strain, which precludes an essential role for any of the mutated proteins in this induction process. On the basis of our observations we propose that the induction signal is related to the number of blocked replication forks: the larger that number, the higher the proportion of induced cells within the population.     

Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome.

Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature.     

New Late Gene, dar, Involved in DNA Replication of Bacteriophage T4 I. Isolation, Characterization, and Genetic Location

Suppressors of gene 59-defective mutants were isolated by screening spontaneous, temperature-sensitive (ts) revertants of the amber mutant, amC5, in gene 59. Six ts revertants were isolated. No gene 59-defective ts recombinant was obtained by crossing each ts revertant with the wild type, T4D. However, suppressors of gene 59-defective mutants were obtained from two of these ts revertants. These suppressor mutants are referred to as dar (DNA arrested restoration). dar mutants specifically restored the abnormalities, both in DNA synthesis and burst size, caused by gene 59-defective mutants to normal levels. It is unlikely that dar mutants are nonsense suppressors since theý failed to suppress amber mutations in 11 other genes investigated. The genetic expression of dar is controlled by gene 55; therefore, dar is a late gene. The genetic location of dar has been mapped between genes 24 and 25, a region contiguous to late genes. dar appears to be another nonessential gene of T4 since burst sizes of dar were almost identical to those of the wild type. Mutations in dar did not affect genetic recombination and repair of UV-damaged DNA, but caused a sensitivity to hydroxyurea in progeny formation. The effect of the dar mutation on host DNA degradation cannot account for its hydroxyurea sensitivity. dar mutant alleles were recessive to the wild-type allele as judged by restoration of arrested DNA synthesis. The possible mechanisms for the suppression of defects in gene 59 are discussed.     

Reversibility of DnaA protein activity in the'irreversible'dnaA204 mutant of Escherichia coli

The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature downshift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42δC. The data indicate that the dnaA204 mutant after a shift to 42δC still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30δC, and after induction of DnaA protein synthesis at 42 C. A model describing the processing of DnaA protein in mutants and in the wild type Is presented.     

Physiological suppression of the temperature-sensitive sporulation defect in a Bacillus subtilis RNA polymerase mutant

Summary Five hundred putative RNA polymerase mutants of Bacillus subtilis were isolated by selecting for resistance to the RNA polymerase inhibitors rifampin (Rif^r), streptovaricin (Str^r) or streptolydigan (Std^r). This collection was screened for mutants that were unable to sporulate at the non-permissive temperature of 46°C, yet which sporulated well at 37°C and had normal vegetative growth (Spo^ts phenotype). Nearly one half of the Rif^r and one quarter of the Stv^r mutants were Spo^ts, whereas none of the Std^r mutants had this phenotype.The streptovaricin resistant strain stv84 was studied in detail. The stv84 mutation maps between cysA14 and strA39 on the B. subtilis chromosome, and the Stv^r and Spo^ts phenotypes cotransform at a frequency of 100%. The Spo^ts phenotype of stv84 could be physiologically corrected by supplementing the growth medium with inhibitors of RNA synthesis such as rifampin or azauracil, with carbohydrates such as ribose, mannose or glycerol, or with lipids such as Tween 40 or fatty acids native to Bacillus subtilis membranes. A Spo^ts phenotype resembling that of stv84 was produced in wild type B. subtilis by adding cerulenin, an inhibitor of fatty acid biosynthesis, to the growth medium. This cerulenin-induced sporulation defect was reversed by the same treatments that correct the temperature-sensitive genetic defect of stv84. These data indicate that the Spo^ts phenotype of strain stv84 is not due to an intrinsic inability of the mutant RNA polymerase to transcribe developmentally-specific genes at the nonpermissive temperature. Rather, the data suggest that the stv84 lesion causes a physiological imbalance which disrupts membrane structure or function in sporulating cells.     

Role of DNA replication in the induction and turn-off of the SOS response in Escherichia coli

Summary We have studied the role of DNA replication in turnon and turn-off of the SOS response in Escherichia coli using a recA::lac fusion to measure levels of recA expression.An active replication fork does not seem to be necessary for mitomycin C induced recA expression: a dnaA43 initiation defective mutant, which does not induce the SOS response at non-permissive temperature, remains mitomycin C inducible after the period of residual DNA synthesis. This induction seems to be dnaC dependent since in a dnaC325 mutant recA expression not only is not induced at 42° C but becomes mitomycin C non-inducible after the period of residual synthesis.Unscheduled halts in DNA replication, generally considered the primary inducing event, are not sufficient to induce the SOS response: no increase in recA expression was observed in dnaG(Ts) mutants cultivated at non-permissive temperature. The replication fork is nonetheless involved in induction, as seen by the increased spontaneous level of recA expression in these strains at permissive temperature.Turn-off of SOS functions can be extremely rapid: induction of recA expression by thymine starvation is reversed within 10 min after restoration of normal DNA replication. We conclude that the factors involved in induction-activated RecA (protease) and the activating molecule (effector)-do not persist in the presence of normal DNA replication.Abbreviations Ts thermosensitive - SDS sodium dodecyl sulfate - Ap ampicillin - UV ultraviolet - X-Gal 5-bromo-4-chloro-3-indolyl--D-galactoside     

Bacillus subtilis RarA acts at the interplay between replication and repair-by-recombination

Bacterial RarA is thought to play crucial roles in the cellular response to blocked replication forks. We show that lack of Bacillus subtilis RarA renders cells very sensitive to H₂O₂, but not to methyl methane sulfonate or 4-nitroquinoline-1-oxide. RarA is epistatic to RecA in response to DNA damage. Inactivation of rarA partially suppressed the DNA repair defect of mutants lacking translesion synthesis polymerases. RarA may contribute to error-prone DNA repair as judged by the reduced frequency of rifampicin-resistant mutants in ΔrarA and in ΔpolY1 ΔrarA cells. The absence of RarA strongly reduced the viability of dnaD23ts and dnaB37ts cells upon partial thermal inactivation, suggesting that ΔrarA cells are deficient in replication fork assembly. A ΔrarA mutation also partially reduced the viability of dnaC30ts and dnaX51ts cells and slightly improved the viability of dnaG40ts cells at semi-permissive temperature. These results suggest that RarA links re-initiation of DNA replication with repair-by-recombination by controlling the access of the replication machinery to a collapsed replication fork.     

Induction of only limited elongation instead of filamentation by inhibition of cell division in Corynebacterium glutamicum

In order to characterize the cell-division mechanism of coryneform bacteria, we tried to isolate cell-division mutants from Corynebacterium glutamicum after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, such as Escherichia coli fts mutants, which form long filaments at the restrictive temperatures. At the non-permissive temperature, most of the mutants formed club-shaped or dumbbell-shaped, elongated rod cells, but no filament formers were isolated. Then we examined the effects of cell division inhibitors on this organism. Cephalexin and sparfloxacin, which are the inhibitors of septation and DNA synthesis respectively, and are known to cause cell filamentation in E. coli, did not cause filamentation in C. glutamicum but induced morphological changes that were similar to those observed with the temperature-sensitive ts mutants of C.␣glutamicum. These results suggest that C. glutamicum has a unique regulation mechanism, that is, the inhibition of cell division leads to cessation of cell elongation. Received: 5 February 1998 / Received revision: 6 April 1998 / Accepted: 27 April 1998     

Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

A DNA replication gene maps near terC in Escherichia coli K12

Summary Temperature-sensitive mutants that filamented at the non-permissive temperature were isolated by specific mutagenesis of the terminus region of the Escherichia coli chromosome. Two of them, mapping at about 35 min, failed to divide due to inhibition of DNA replication. Further characterization indicated that these mutants are temperature-sensitive for DNA chain elongation.     

Alteration of the rate of cell division independent of the rate of DNA synthesis in a mutant of Salmonella typhimurium

Summary Salmonella typhimurium strain IIG has a temperature—sensitive DNA synthesis initiation apparatus and completes rounds of DNA replication when shifted to 38°. At this temperature there is a period of apparently normal division followed by a second phase in which DNA-less cells are produced. The rate of division in this second phase can be markedly increased if a culture growing in MM is shifted to nutrient broth at the time of the temperature shift. The extra divisions induced by the nutritional shift are not due to extra replication forks being introduced by this process nor to the rapid growth of ts ⁺ revertants. It is concluded that in this strain at 38°, the rate of division can be increased without altering the rate of DNA synthesis. The extra divisions induced by the shift-up do not take place for about 90 min. The possible occurrence of such a period between the triggering of division and the division event in normal cells is discussed.