High frequency of genetic duplications in the dnaB region of the Escherichia coli K12 chromosome.

The region that includes the dnaB locus on the E. coli K12 chromosome was shown to be duplicated at high frequency in cell populations. The duplications were shown to be arranged in tandem and segregated at various frequencies. Segregation was dependent on the recA recombination system, but independent of recB,C. Though most of the data was obtained with dnaB::Tn10 insertion mutants, the duplications were shown to occur in the absence of Tn10.

dnaA alleles are recessive.

Dominance tests of several dnaA alleles from Escherichia coli, including two previously reported to be dominant, show that all of the mutant alleles examined are recessive to dnaA+.

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.

Characterization of arithmetic deoxyribonucleic acid synthesis at restrictive temperature in a dnaE mutant of Escherichia coli K-12.

The dna-293 mutation is shown to be a dnaE allele. The linear deoxyribonucleic acid synthesis previously observed in this mutant has been further characterized. The production of small deoxyribonucleic acid intermediates and their subsequent joining were identical in the mutant and its dnaE+ parent at 42.5 degrees C. Though the mutant cells continued to divide at the nonpermissive temperature, the rate of division was reduced. The data are consistent with a lack of production of replicationally active deoxyribonucleic acid polymerase III at the restrictive temperature.

Dominance of dnaA+ to dnaA in Escherichia coli.

The dominance of dnaA+ to the dnaA508 mutation was complete and was unaffected by the presence of a copy of the chromosomal replication origin on the episome. These results prove that the dnaA gene of Escherichia coli produces a diffusible product.

Antipolarity in the ilv operon of Escherichia coli K-12.

The genes governing three of the enzymes of the isoleucine-valine biosynthetic pathway form the operon: operator-ilvA-ilvD-ilvE. The enzymes are: ilvA, l-threonine deaminase; ilvD, dihydroxy acid dehydrase; and ilvE, transaminase B. A nonsense mutation in the ilvD gene (D-ochre) and a nonsense mutation in the ilvE gene (E-amber) affect the properties of the proximal gene product, l-threonine deaminase (TD), in addition to inactivating the enzymes produced by the genes in which the mutations have occurred. The D-ochre mutation causes TD to move in diffusion and gel filtration experiments as though it were 30% smaller than the wild-type enzyme. The E-amber mutation causes TD to move in similar experiments as though it were much larger than the wild-type enzyme. Both mutations completely abolish the sensitivity of TD to l-isoleucine, the normal feedback inhibitor of the wild-type enzyme. The effects of the nonsense mutations on TD can be reversed in three ways: by genetic reversion of the D-ochre mutation; by treatment of the altered enzymes with 3.0 m urea; and by forming a heterozygous diploid, containing the wild-type allele as well as the mutant allele of ilvD or ilvE. The results suggest that the subunits of TD undergo abnormal aggregation in the presence of the partial polypeptides produced by the mutant alleles of ilvD or ilvE; multi-enzyme aggregates in extracts of wild type, however, could not be detected.

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.

Complementation of a dnaC initiation defect in vitro.

The dnaC28 mutant, CT28-3b, is an initiation defective dnaC strain. Extracts of the mutant failed to synthesize DNA in vitro when the strain was incubated at the restrictive temperature for two generation times prior to preparation of the extract. Addition of a complementing extract from a Col-E1::dnaC+ hybrid plasmid containing strain or of partially purified dnaC protein resulted in substantial synthesis. Hybridization of the DNA made by these in vitro complementation extracts showed that a significant portion of this DNA was from the region near the chromosomal origin of replication.

RNA polymerase is required for DNA initiation in vitro.

We have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA. To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used. Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA polymerase antibody abolished ribonucleic acid synthesis completely. Antibody effectively inhibited chromosome initiation in the dnaA mutant based reaction but streptolydigin did not. Neither streptolydigin nor antibody affected the dnaC-dependent assay. It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide. A scheme for the sequence of initiation events is presented.

Suppression of dnaC alleles by the dnaB analog (ban protein) of bacteriophage P1.

The dnaB analog protein produced by the ban gene of bacteriophage P1 was shown to suppress several Escherichia coli dnaC alleles. Suppression of dnaC7 temperature sensitivity in P1 lysogens of a dnaC7 mutant was complete at all temperatures. For the dnaC2 and dnaC28 alleles, suppression was observed only at intermediate temperatures. Though these intermediate temperatures were sufficient to completely restrict the mutants, at higher temperatures the suppression was not observed. No suppression of the dnaC1 allele was detected. These results have implications concerning the requirement for the dnaB-dnaC complex at the various stages of deoxyribonucleic acid replication.

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.

Isolation and analysis of multicopy extragenic suppressors of dnaA mutations.

Recombinant plasmids were constructed from restriction enzyme digests of Escherichia coli chromosomal deoxyribonucleic acid and pMB9 plasmid deoxyribonucleic acid and selected for correction of the dnaA phenotype. The three plasmids isolated, all retransformed dnaA cells, both recA+ and recA, such that all tetracycline-resistant transformants selected at permissive temperature simultaneously became temperature resistant. Restriction enzyme mapping of the plasmids showed all three to be different, and it was subsequently shown that none contained the dnaA+ gene. Though each of the three plasmids suppressed three different temperature-sensitive dnaA alleles, none corrected the phenotype of an unsuppressed dnaA amber allele. It was concluded, therefore, that each plasmid contained a unique extragenic suppressor of dnaA and that the suppression was observed because of the elevated gene dosage of the cloned material. The plasmids were unstable in the absence of selection.

RNA polymerase interaction with dnaB protein and lambda P protein during lambda replication.

The Escherichia coli GroP- phenotype, associated with some dnaB mutants and measured as a decreased ability to plate lambda bacteriophage, was altered by some rpoB mutations. The rpoB effect showed an allele specificity. The participation both of dnaB and of lambda P alleles in the GroP- phenotype was also allele specific. It was concluded that RNA polymerase, dnaB protein, and lambda P protein form a functional complex required for lambda replication.

Initiation of chromosomal DNA synthesis in vitro.

An in vitro complementation assay for initiation of chromosomal DNA replication is described. The initiation reaction is dependent upon extract from either of two hybrid-plasmid containing strains. Each hybrid plasmid carries a suppressor of dna A-ts mutations. The in vitro DNA synthesis is heavily biased toward the origin region, and the origin of replication (oriC) is replicated as determined by DNA-DNA hybridizations.

DNA replication intermediates synthesized by lysates of dnaB, dnaG and dnaB dnaG mutants in vitro.

Isogenic dnaB, dnaG, and dnaB dnaG mutants were constructed and used as extracts in the cellophane-disc in vitro DNA replication system. The increased proportion of 5S DNA characteristics of the dnab extract and the lack of Okazaki piece synthesis characteristic of the dnaG extract were both apparent in analysis of the dnaB dnaG mutant extract reaction. A hypothetical scheme to explain these results and those of others is presented.

The isolation and characterization of escherichia coli dnaB::Tn10 insertion mutations.

Exploitation of the ability of the ban protein encoded by phage P1 to compensate for dnaB-defective host mutations, allowed the isolation of dnaB::Tn10 insertion mutations. The presence of P1bac prophage was required for survival of dnaB::Tn10 mutants, and such lysogens were cryosensitive. The insertions were shown to map in dnaB by transduction and this was confirmed by complementation analysis. The dnaB::Tn10 (P1bac) strains were non-permissive for lambda growth but did support the growth of lambda-dnaB+ specialized transducing phage. No antigenically active dnaB product could be detected by immunologic assays using either of two methods. In addition, it was shown that the observe cryosensitivity of P1bac suppression was a direct result of reversible inactivation of the ban protein at low temperature.

Analysis of DNA polymerases II and 3 in mutants of Escherichia coli thermosensitive for DNA synthesis.

A series of double mutants carrying one of the thermosensitive mutations for DNA synthesis (dnaA, B, C, D, E, F, and G) and the polA1 mutation of DeLucia and Cairns, were constructed. Enzyme activities of DNA Polymerases II and III were measured in each mutant. DNA Polymerase II activity was normal in all strains tested. DNA Polymerase III activity is thermosensitive specifically in those strains having thermoscnsitive mutations at the dnaE locus. From these results we conclude that DNA Polymerases II and III are independent enzymes and that DNA Polymerase III is an enzyme required for DNA replication in Escherichia coli.

Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication.

Thermosensitive deoxyribonucleic acid replication-defective mutants have been isolated by using an autoradiographic selection method. The mutants have been analyzed genetically and biochemically. Some of the mutants show thermosensitivity of in vitro deoxyribonucleic acid replication. These can be classified into three groups according to their behavior in in vitro complementation assays. This classification is congruent with that obtained by genetic mapping by using cotransduction frequencies with selected markers in P1 transduction analysis.