NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication.

The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E. coli HfrHl65/120/6 dna-252. This dna mutation was incorrectly classed as a dnaA mutation. Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants. Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied. Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to cholramphenicol. Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA). Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an orgin-RNA species. These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process.     

Genetic and phenotypic characterization of dnaC mutations.

The dna-1, dna-2, dna-7, and dna-28 mutations, all of which are located near min 89.5 on the E. coli linkage map, have been characterized further. As previously demonstrated for dna-2 and dna-28, neither the dna-1 nor dna-7 mutation affects the ability of a strain to produce bacteriophage lambda at temperatures non-permissive for the continued replication of the bacterial chromosome. The reported temperature-sensitive inhibition of lambda production in a strain carrying dna-7 is shown to be a consequence of a thermosensitive host specificity mutation in the hsm gene and not of the dna-7 mutation. The four dna mutations are recessive to the wild type and define a single dnaC cistron according to standard complementation criteria. Unlike other characterized dnaC mutants, however, strains carrying the dnaC1 or dnaC7 alleles exhibit an abrupt cessation of deoxyribonucleic acid synthesis at 42 C that appears to be more compatible with a defect in deoxyribonucleic acid chain elongation rather than in initiation. The possibility that the apparent elongation defect is actually a composite effect of residual synthesis and deoxyribonucleic acid degradation is raised by the net deoxyribonucleic acid degradation observed in the dnaC1 strain at 42 C. Several alternative possibilities for the function of the dnaC gene product are suggested.     

Prophage Induction by High Temperature in Thermosensitive dna Mutants Lysogenic for Bacteriophage Lambda

High-temperature treatment of thermosensitive dna mutants lysogenic for phage lambda leads to prophage induction and release of phage (at the permissive temperature) in elongation-defective mutants of the genotypes dnaB, dnaE, and dnaG. In initiation-defective mutants no prophage induction occurs at 42 C in mutants of the genotype dnaA, whereas with a dnaC mutant as well as with strain HfrH 252 (map position not yet known) phages are released at 42 C. DNA degradation at the replication fork at 42 C is observed in all dnaB(lambda) mutants tested, but not in mutants of the genotypes dnaE(lambda) and dnaG(lambda). Therefore, degradation of replication fork DNA is not a prerequisite for prophage induction.     

Bacteriophage G4 DNA synthesis in temperature-sensitive dna mutants of Escherichia coli.

The synthesis of bacteriophage G4 DNA was examined in temperature-sensitive dna mutants under permissive and nonpermissive conditions. The infecting single-stranded G4 DNA was converted to the parental replicative form (RF) at the nonpermissive temperature in infected cells containing a temperature sensitive mutation in the dnaA, dnaB, dnaC, dnaE, or dnaG gene. The presence of 30 mug of chloramphenicol or 200 mug of rifampin per ml had no effect on parental RF synthesis in these mutants. Replication of G4 double-stranded RF DNA occurred at a normal rate in dnaAts cells at the nonpermissive temperature, but the rate was greatly reduced in cells containing a temperature-sensitive mutation in the dnaB, dnaC, dnaE, or dnaG gene. RF DNA replicated at normal rates in revertants of these dna temperature-sensitive host cells. The simplest interpretation of these observations is that none of the dna gene products tested is essential for the synthesis of the complementary DNA strand on the infecting single-stranded G4 DNA, whereas the dnaB, dnaC, dnaE, (DNA polymerase III), and dnaG gene products are all essential for replication of the double-stranded G4 RF DNA. The alternate possibility that one or more of the gene products are actually essential for G4 parental RF synthesis, even though this synthesis is not defective in the mutant hosts, is also discussed.     

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.     

Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.     

The Escherichia coli dnaJ mutation affects biosynthesis of specific proteins, including those of the lac operon.

Temperature-sensitive dnaJ mutants of Escherichia coli showed a thermosensitive defect in the synthesis of beta-galactosidase. Synthesis of the lac mRNA was greatly reduced at the restrictive temperature. The mutants were also conditionally defective in the synthesis of a subset of membrane proteins such as succinate dehydrogenase, whereas the synthesis of anthranilate synthetase, encoded by trpED, as well as that of most cellular proteins, was unaffected at the restrictive temperature. The defect was specific for the dnaJ mutants among several dna mutants which are known to be involved in the initiation of DNA synthesis: dnaK, dnaA, and dnaB mutants synthesized each of these proteins normally even at the restrictive temperature. At the restrictive temperature, growth of the dnaJ mutants was arrested at a specific stage of the cell cycle.     

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.     

Mutants of Escherichia coli with Cold-Sensitive Deoxyribonucleic Acid Synthesis

Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.     

Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.     

The dnaB-dnaC replication protein complex of Escherichia coli. II. Role of the complex in mobilizing dnaB functions

The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions. Within the dnaB-dnaC complex, dnaB appears to be inactive but becomes active upon the ATP-dependent release of dnaC from the complex. With adenosine 5'-(gamma-thio)triphosphate substituted for ATP, the dnaB-dnaC complex does not direct dnaB to its targeted actions. Excess dnaC inhibits dna beta actions and augments the ATP gamma S effects. In the dnaA protein-driven initiation of duplex chromosome replication, dnaB is introduced for its essential helicase role via the dnaB-dnaC complex. Similarly, when the dnaA protein interacts nonspecifically with single-stranded DNA, the dnaB-dnaC complex is essential to introduce dnaB for its role in primer formation by primase.     

Pleiotropic, extragenic suppression of dna mutants in Bacillus subtilis.

Thermosensitive (dna) mutants of Bacillus subtilis defective in deoxyribonucleic acid replication can be divided into two groups on the basis of their ability to spontaneously yield secondary mutants with an HDS phenotype (thermoin-sensitivity and resistance to aryl-azo-pyrimidines) at frequencies higher than 10(-8). Such a phenotype is due to alleles at the hds locus (mapping close to cysA), which act as extragenic pleiotropic suppressors. HDS suppressibility has been used as a screening tool to identify new dna strains among uncharacterized temperature-sensitive mutants.     

Replication of G4 progeny double-stranded DNA in dna mutants of Escherichia coli.

Host functions required for replication of progeny double-stranded DNA of bacteriophage G4 were examined by using metabolic inhibitors and Escherichia coli dna mutants. In dna+ bacteria, synthesis of the progeny replicative form (RF) was relatively resistant to 30 microgram/ml of chloramphenicol, but considerably sensitive to 200 microgram/ml of rifampicin. The RF replication was severely inhibited by 50 microgram/ml of mitomycin C, 50 microgram/ml of nalidixic acid, or 200 microgram/ml of novobiocin. At 41 degrees C, synthesis of G4 progeny RF was distinctly affected in a dnaC(D) mutant and in a dnaG host. The progeny RF replication was prevented at 42 degrees C in a dnaE strain as well as in a dnaB mutant. In a dnaZ strain, the synthetic rate of the progeny RF was markedly reduced at 42 degrees C. At 43 degrees C, the rate of G4 progeny RF synthesis was reduced even in dna+ or dnaA bacteria, but significant amounts of the progeny RF were still synthesized in these hosts at the high temperature. In addition to five dna gene products, host rep function was essential for the RF replication.     

Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants

Abstract The temperature-sensitive dna mutants of the budding yeast Saccharomyces cerevisiae (Dumas et al. (1982) Mol. Gen. Genet. 187, 42–46) are more inhibited in DNA synthesis than in protein synthesis. These properties are also characteristics of many yeast mutations that inhibit progress through the cell cycle. Therefore we surveyed the collection of dna mutants for cell-cycle mutations. By genetic complementation we found that dna 1 = cdc 22, dna 6 = cdc 34, dna 19 = cdc 36, and dna 39 = dbf 3. Furthermore, by direct gene cloning we found that the dna26 mutation is allelic to prt1 mutations, which are known to exert primary inhibition on protein synthesis. This protein-synthesis mutation exerts a dna phenotype due to cell-cycle inhibition: prt1 mutations can block the regulatory step of the cell cycle while allowing significant amounts of protein synthesis to continue. Our non-exhausive screening suggests that the dna mutants may house other mutations that affect the yeast cell cycle.     

Rifampin disrupts conjugal and chromosomal deoxyribonucleic acid metabolism in Escherichia coli K-12 carrying some IncIalpha plasmids.

The effects of rifampin and chloramphenicol on the transfer of ColIdrd-1 have been examined to determined whether transfer requires the synthesis of an untranslated species of ribonucleic acid (RNA), as proposed in models for the transfer of another IncIalpha plasmid, R64drd-11. When RNA synthesis was inhibited throughout mating by rifampin, ColI transfer between dna+ bacteria occurred at the normal rate for about 10 min and then stopped abruptly. Conjugational deoxyribonucleic acid (DNA) synthesis in dnaB mutants indicates that plasmid DNA was made in the rifampin-treated donors to replace the transferred material but the DNA tended to be unstable. In the presence of chloramphenicol, transfer of ColI gradually diminished over a longer period. Rifampin, but not chloramphenicol, was found to have unpredicted effects on chromosomal DNA metabolism in unmated dna+ and dnaB bacteria when they harbor any of three IncIalpha plasmids (ColIdrd-1, R144drd-3, and R64drd-11). Replication of the bacterial chromosome in such cells stopped abruptly about 15 min after the addition of rifampin, and at 41 degrees C, but not 37 degrees C, this was followed by extensive DNA breakdown. These findings suggest that curtailment of IncIalpha plasmid transfer by the drug results from a general disruption of DNA metabolism rather than from inhibition of a species of RNA essential for transfer.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

Completed Bacillus subtilis nucleoid as a doublet structure.

When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-1, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)-less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were foud to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.     

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.