Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein.

The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.     

Genetic Mapping of a Group of Temperature-Sensitive dna Initiation Mutants in BACILLUS SUBTILIS

Recombination frequencies among temperature-sensitive dna mutants from various laboratories were analyzed, and eleven dna mutants were found to be closely linked. They are classified as group B dna mutants, since these are closely linked with dnaB19, originally isolated and approximately mapped near leuA8 by KARAMATA and GROSS (1970). However, the dnaB19 mutation itself has relatively high recombination frequencies with the other mutations, thus, we propose to subdivide the dnaB group into two subgroups--dnaBI, including ten mutants (dna-1, dna-3, dna-5, dna-17, dna-27, dna-51, dna-60, dna-62, dna-103 and dna-134) and dnaBII, including dnaB19. The map order of dnaB and markers in the vicinity was determined to be argA-citH-citC-phoP-PhoR-polA-dnaBI-dnaBII-citF-leuA-pheA.     

Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage.

The Escherichia coli dnaB252 allele is the only dnaB mutation which confers a deoxyribonucleic acid initiation-defective phenotype on the cell. The presence of a multicopy hybrid plasmid containing the dnaC+ gene in a dnaB252 strain completely suppressed the temperature-sensitive phenotype. It is suggested that at high temperature the dnaB252 protein has a lowered affinity for dnaC protein, and that the formation of a dnaB-dnaC complex is mandatory for initiation.     

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.     

Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.     

dnaB125, a dnaB nonsense mutation

A temperature-sensitive dnaB mutation, dnaB125, was shown to be a suppressed amber mutation. The effects of inserting different amino acids at the mutated site via amber suppressors were examined for both Escherichia coli and bacteriophage gamma growth. In addition, the dnaB125 amber allele was shown to be different from the previously described dnaB amber allele, dnaB266. The extent of residual deoxyribonucleic acid synthesis observed in a supF(Ts) dnaB125 strain at high temperature revealed that the dnaB protein was present in excess and that deoxyribonucleic acid synthesis could continue for several generation equivalents without further production of dnaB protein.     

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor.

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.     

Identification of the product of dnaB gene in Bacillus subtilis

In order to detect the product of dnaB gene in B. subtilis, a gene which is involved in the initiation of DNA replication and the formation of the DNA-membrane complex, we synthesized an origopeptide of 15 amino acids which corresponds to a region near the carboxyl-terminal of the gene product, and raised antibody against the synthetic peptide. We have also employed a filter binding assay to measure the predicted DNA binding activity of the product of the dnaB gene, using the plasmid pUB110. The binding activity was detected after fractionation of cell lysates of B. subtilis on sucrose-density gradients. When the active fraction was prepared from a mutant which was temperature-sensitive for the dnaB gene, the DNA binding activity in the fraction showed significant thermolability. Furthermore, the binding activity was inhibited by the purified antibody raised against the synthetic peptide. These results suggest that the product of the dnaB gene does indeed have DNA binding activity, and that the filter binding assay and the antibody can be used for the detection and characterization of the gene product.     

Initiation of Deoxyribonucleic Acid Replication in Germinating Spores of Bacillus subtilis 168 Carrying the dnaB(Ts)134 Mutation

The nature of the deoxyribonucleic acid synthesis reported by others to occur at 45 degrees C in germinating spores of the temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of B. subtilis 168, one carrying and the other without the dnaB (Ts)134 mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 45 degrees C. At a slightly higher temperature, 49 degrees C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts(+) strain. Thus, it is concluded that, after the germination of a spore, the action of the dnaB134 gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 45 degrees C is presumably due to incomplete inactivation of the dnaB134 gene product under such conditions.     

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.     

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.     

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.     

On the participation of RecB in the induction of mini-Tn10 precise excision in a dnaB thermosensitive mutant

Precise excision of transposons Tn10 and mini-Tn10 is increased in the dnaB252 thermosensitive mutant of Escherichia coli K12, at the permissive temperature. DNA repair proteins like Pol II, RecF, Ruv and RecA were found to participate, to different extents, in this induced excision event. In this work we report that DNA repair-recombination protein RecBCD has a predominant role in this deletion process. The role of this and other repair proteins in DNA replication of the dnaB mutant in relation to the excision of the transposon is analyzed.     

dfp Gene of Escherichia coli K-12, a locus affecting DNA synthesis, codes for a flavoprotein.

The cloned dfp gene complements dna-707 (now designated dfp-707), a temperature-sensitive conditionally lethal mutation that results in a slow cessation of DNA synthesis while protein synthesis is maintained. In vitro and in vivo experiments failed to demonstrate a specific defect in the initiation of DNA replication, and turn-off of DNA synthesis at high temperature was slower than that of a typical initiation (dnaA) mutant. The gene was localized, and its product was identified through the construction and analysis of deletion and insertion mutants of dfp-containing plasmids. dfp is located between the rpmB and dut genes at 81 min on the linkage map of Escherichia coli K-12. It is transcribed clockwise, independently of dut. The ability of a plasmid to complement a chromosomal dfp-707 mutation was correlated with its ability to produce a 45-kilodalton polypeptide. The purified protein contained 1 mol of flavin mononucleotide per mol of polypeptide.     

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.     

Thymineless death in Escherichia coli dnaB mutants and in a dnaB dnaG double mutant.

The interference of dnaB mutations of Escherichia coli with thymineless death is described. All the isogenic Thy- dnaB mutants of E. coli we have tested show a remarkable immunity towards cell death induced by thymine deprivation at the nonpermissive temperature. We have also constructed and tested an isogenic double dnaB dnaG mutant. It loses its viability in the absence of thymine at both permissive and nonpermissive temperatures. The role of the dnaB gene product is discussed.     

Analogs of the dnaB gene of Escherichia coli K-12 associated with conjugative R plasmids.

The dnaB266(Am) mutation in Escherichia coli K-12 is an amber mutation such that strains carrying this mutation are not viable in a sup+ strain. With five different R plasmids, it has been possible to construct viable R+ derivatives of this amber mutant and show that the plasmids themselves do not carry amber suppressors. This is interpreted as evidence for the presence of dnaB analog genes associated with these plasmids. Plasmid-positive strains carrying these genes often showed some degree of cryosensitivity of DNA synthesis and colony-forming ability. These observations indicate that the presence of dnaB analog genes in association with R plasmids must be relevant to the plasmid state or to some aspect of conjugative ability.     

Dominant lethal mutations in the dnaB helicase gene of Salmonella typhimurium

A class of dominant lethal mutations in the dnaB (replicative helicase) gene of Salmonella typhimurium is described. The mutated genes, when present on multicopy plasmids, interfered with colony formation by Escherichia coli host strains with a functional chromosomal dnaB gene. The lethal phenotype was expressed specifically in supE (glutamine-inserting) host strains and not in Sup+ strains, because the mutant genes, by design, also possessed an amber mutation derived from a glutamine codon. Mutations located at 11 sites by deletion mapping and DNA sequence analysis varied in the temperature dependence and severity of their lethal effects. None of the mutations complemented a dnaB(Ts) host strain at high temperature (42 degrees C). Therefore, these nonfunctional DnaB proteins must engage some component(s) of the DNA replication machinery and inhibit replication. These mutations are predicted to confer limited, specific defects in either the catalytic activity of DnaB or the ability of DnaB to interact with one of its ligands such as DNA, nucleotide, or another replication protein. The variety of mutant sites and detailed phenotypes represented in this group of mutations may indicate the operation of more than one specific mechanism of lethality.