DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect.

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.     

Different effect of mutations in Escherichia coli K12 dna-genes on the transposition of Tn5- and Tn10-elements

The effect of mutations in dnaA(dnaA46), dnaG(dnaG3), dnaC (dnaC1 and dnaC2) and dnaB genes on transposition of two transposons, Tn5 and Tn10, from bacteriophage lambda genome into the chromosome of host cells has been studied. Transposition was performed at permissive temperatures for the mutant recipients. The mutations in dnaA, dnaC, dnaG genes were shown to decrease the transposition of Tn10 for some orders of magnitude as compared with transposition registered in wild type cells. Independence of Tn5 transposition of the above mentioned genes was demonstrated, providing evidence on the different modes of transposition of these two Tn-elements.     

Interactions of DNA replication factors in vivo as detected by introduction of suppressor alleles of dnaA into other temperature-sensitive dna mutants.

Suppressor mutations located within dnaA can suppress the temperature sensitivity of a dnaZ polymerization mutant, indicating in vivo interaction of the products of these genes. The suppressor allele of dnaA [designated dnaA(SUZ, Cs)] could not be introduced, even at the permissive temperature, by transduction into temperature-sensitive (Ts) dnaC or dnaG recipients; it was transduced into dnaB(Ts) and dnaE(Ts) strains but at very low frequency. Recipient cells which were dnaA+ dnaE(Ts) were killed by the incoming dnaA(SUZ, Cs) allele, and it is presumed that combinations of dnaA(SUZ, Cs) with dnaB(Ts), dnaC(Ts), or dnaG(Ts) are lethal also. In one specific case, the lethality required the presence of three alleles: the incoming dnaA suppressor mutation, the resident dnaA+ gene, and the dnaB(Ts) gene. This was shown by the fact that dnaB(Ts) could readily be introduced into a dnaA(SUZ, Cs) dnaB+ recipient. That is, in the absence of dnaA+, the dnaA suppressor and dnaB(Ts) double mutant was stable. One model to explain these results proposes that the dnaA protein functions not only in initiation but also in the replication complex which contains multiple copies of dnaA and other replication factors.     

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.     

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.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

dnaA acts before dnaC in the initiation of DNA replication

We constructed a double mutant of Escherichia coli K-12 carrying dnaA(Ts) and dnaC(Cs) lesions. In this mutant DNA synthesis proeceeds normally at 32 degrees C and initiation is inhibited at both 41 and 20 degrees C. By shifting this culture grown at 32 degrees C to the two restrictive temperatures in different time sequences and assaying protein and DNA synthesis of cells growing at different temperatures, we found that dnaA and dnaC genes work independently with dnaA acting before dnaC. While preparing special strains for this work, we also showed that the order of genes in the neighborhood of dnaA is dnaA-tnaA-phoS-ilv.     

In vitro assembly of a prepriming complex at the origin of the Escherichia coli chromosome

During initiation of DNA replication of plasmids containing the origin of the Escherichia coli chromosome (oriC), the proteins dnaA, dnaB, and dnaC interact and assemble a complex at oriC. The complex is larger and more asymmetric than that formed by dnaA protein and embraces an extra 50 base pairs at the left side of the minimal oriC sequence. Both dnaA and dnaB proteins have been identified in the complex by electron microscopy and antibody binding; dnaC protein was not detected. HU protein, which stimulates the activity of the initiation reaction, was often present. Entry of dnaB protein required dnaA and dnaC proteins and a supercoiled template. Thus, a complex structure, involving multiple proteins and a large region of DNA, must be formed at the origin to prepare the template for priming and replication.     

Initiation of chromosome replication in dnaA and dnaC mutants of Escherichia coli B/r F.

Regulatory aspects of chromosome replication were investigated in dnaA5 and dnaC2 mutants of the Escherichia coli B/r F. When cultures growing at 25 degrees C were shifted to 41 degrees C for extended periods and then returned to 25 degrees C, the subsequent synchronous initiations of chromosome replication were spaced at fixed intervals. When chloramphenicol was added coincident with the temperature downshift, the extend of chromosome replication in the dnaA mutant was greater than that in the dnaC mutant, but the time intervals between initiations were the same in both mutants. Furthermore, the time interval between the first two initiation events was unaffected by alterations in the rate of rifampin-sensitive RNA synthesis or cell mass increase. In the dnaC2 mutant, the capacities for both initiations were achieved in the absence of extensive DNA replication at 25 degrees C as long as protein synthesis was permitted, but the cells did not progress toward the second initiation at 25 degrees C when both protein synthesis and DNA replication were prevented. Cells of the dnaA5 mutant did not achieve the capacity for the second initiation event in the absence of extensive chromosome replication, although delayed initiation may have taken place. A plausible hypothesis to explain the data is that the minimum interval is determined by the time required for formation of a supercoiled, membrane-attached structure in the vicinity of oriC which is required for initiation of DNA synthesis.     

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.     

Host requirements for growth of lambda-P22 hybrid in Escherichia coli.

The requirements for growth of bacteriophage lambda containing the deoxyribonucleic acid replication region from Salmonella phage P22 were determined in a burst size experiment. The products of genes dnaE, dnaJ, dnaK, dnaY, dnaZ, and seg were required, but not the products of genes dnaA, dnaB, dnaC, and dnaX. This lambda-P22 hybrid phage was also dependent on polA for growth at 32 degrees C.     

Plasmid ColE1 DNA replication in Escherichia coli strains temperature-sensitive for DNA replication

Mutants of the dnaA, dnaC, dnaD, polC, dnaF and dnaG gene loci were tested for their capacity for colicinogenic plasmid E1 (ColE1) replication at a non-permissive temperature. It was found that ColE1 replication was independent of the dnaA gene function and dependent on dnaC, D, F and G. ColE1 replication in the polC mutant E486 continued for several hours but at a greatly reduced rate. No effect was found of the dnaG mutation on thymine-deprivation-induced "priming" of ColE1 replication at the non-permissive temperature. The mutants also were tested for aberrant replication intermediates of plasmid DNA as well as a temperature sensitive supercoiled DNA-protein relaxation complex. RNA-containing supercoils were found to accumulate in a poIC mutant also blocked for protein synthesis.     

Signal of induction of recA protein in E. coli

The nature of the signal(s) responsible for the induction of the SOS functions in E. coli was investigated in dnaA and dnaC mutants, in which recA protein was induced by UV irradiation under conditions where no DNA replication could occur. This induction was dependent upon an active excision-repair system, since it was abolished in a dnaC uvrB double mutant at non-permissive temperature. In such a case, the addition of bleomycin, an agent known to produce single-strand breaks into DNA, was able to restore the induction of the recA protein.     

A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene

Summary An 8.2 kb fragment of E. coli chromosomal DNA, when cloned in increased copy number, suppresses the dnaA46 mutation, and an abundant protein of about 68 kd (60 kd when measured by us), encoded by the fragment, is essential for the suppression (Takeda and Hirota 1982). Mapping experiments show that the fragment originates from the 94 min region of the chromosome. It encodes several proteins but only one abundant polypeptide of the correct size, the product of the groEL gene. Suppression by the fragment is allele specific; those mutations which map to the centre of the gene are suppressed. Other initiation mutants including dnaA203, dnaA204, dnaA508, dnaAam, dnaC, dnaP and dnaB252 are not suppressed. Most suppressed strains are cold-sensitive suggesting an interaction between the mutant proteins (or their genes) and the suppressing protein or proteins.     

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.     

Timing of chromosomal replication in Escherichia coli

We have previously shown that certain mutations in the dnaA and recA genes of Escherichia coli perturb initiation of chromosomal replication so that all origins present are not initiated simultaneously. In this work, several genes whose protein products are involved in initiation of replication have been investigated for their effects on the synchrony of initiation. Some of the mutants (dnaC2, rpoC907, dam3) were found to have the asynchrony phenotype. Also, dnaA(Ts) mutations were shown to be dominant over dnaA+ in terms of initiation synchrony. The mechanism leading to the asynchronous phenotype is discussed.     

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.     

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.     

Inhibitors of DNA synthesis cause excessive DNA synthesis in dnaA mutants of Escherichia coli K12.

The relative rate of net DNA synthesis was stimulated when cells of dnaA mutants of Escherichia coli K12 were grown in the presence of low concentrations of DNA synthesis inhibitors. This led to a supernormal DNA/cell mass ratio. The excessive DNA was similar to the normal chromosomal DNA in size and stability in vivo. However, the cells did not divide but turned into long filaments. Excessive DNA synthesis in the presence of inhibitors of DNA synthesis was observed in the cultures of two independent dnaA mutants of E. coli, but dnaB and dnaC mutants behaved like the wild type in this respect.     

Growth of Salmonella bacteriophage P22 in Escherichia coli dna(Ts) mutants.

Salmonella bacteriophage P22 grows in two deoxyribonucleic acid initiation mutants of Escherichia coli under nonpermissive conditions, dnaA and dnaC. Functional products of genes dnaE, dnaZ, lig, dnaK, and dnaG are indispensable for deoxyribonucleic acid replication of P22. In 11 E. coli dnaB mutants belonging to all phenotypic groups, phage were produced at 42 degrees C.