Initation of DNA replication in Escherichia coli. I. Characteristics of the initation process in dna mutants

Summary When a culture of E. coli strain carrying a temperature-sensitive DNA initiation mutation, dna-167 or dnaC2, is exposed to a nonpermissive temperature for a certain period of time, and then transferred back to a permissive temperature, DNA synthesis is resumed even in the presence of chloramphenicol. This shows that thermolabile components coded by either of these mutated genes can be reactivated after return to permissive temperatures, and consequently initiation of a new replication cycle can occur in the absence of concomitant protein synthesis in both strains. The reinitiation of replication occurring after lowering the temperature is sensitive to rifampicin in the dna-167 cells, but not in the dnaC2 mutant. The capacity for initiating a new round of replication is very labile in the dna-167 mutant, but not in the dnaC2 mutant, when a culture of the mutant is maintained at a nonpermissive temperature in the presence of rifampicin. Mechanisms of blocking of the initiation process with these mutants are discussed.After a prolonged exposure of an early-exponential phase culture to high temperatures, reinitiation of DNA replication never exceeds a doubling in both strains, when the temperature is lowered in the presence of chloramphenicol. However, after an exposure of a late-exponential phase culture to a nonpermissive temperature, more than one round of replication occurs in both strains even in the presence of chloramphenicol.     

Replication of Fpoh+ plasmid in mafA mutants of Escherichia coli defective in plasmid maintenance

Summary A class of F plasmids, designated Fpoh ⁺, was previously shown to be able to replicate extrachromosomally on Hfr strains by virtue of carrying the specific site or region poh ⁺ (permissive on Hfr) of the E. coli chromosome (Hiraga, 1975, 1976a). These plasmids were now found to replicate on E. coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids. The derivatives of Fpoh ⁺ that have lost the poh ⁺ site, on the other hand, failed to replicate on mafA mutants. These mutants harboring Fpoh ⁺ (but not Poh^- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh ⁺ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons. It is tentatively concluded that the poh ⁺ site is required for F plasmids to replicate on mafA mutants as well as on Hfr strains. In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh ⁺ region contains the replication origin of the E. coli chromosome.     

Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).     

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.     

Plasmid replication and Hfr formation in strains of Escherichia coli carrying seg mutations.

Summary Several conditional-lethal mutations that do not permit the replication of F-factors ofEscherichia coli K-12 are located at a site calledseg. This gene is located on theE. coli chromosome betweenserB andthr. It is unrelated to other known genes involved in DNA replication. Strains carryingseg mutations were unable to replicate F-lac⁺, several F-gal⁺s, F-his⁺ and bacteriophage at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C.When the kinetics of the loss of F-primes is studied inseg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F⁺ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F⁺ state did not show F-genote formation.F-genote formation fromseg Hfr strains is dependent of a functionalrecA gene, as F-genote formation was not seen with aseg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F-gal⁺, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.     

The Characteristics and Genetic Map Location of a Temperature Sensitive DNA Mutant of E. COLI K12

A temperature sensitive strain of E. coli K12 has been isolated in which residual DNA synthesis occurs at the 40 degrees C restrictive temperature; syntheses of RNA, protein and DNA precursors are not directly affected. The mutation has been designated dna-325 and is located at 89 min on the E. coli map in the same region where the dnaC locus is found. dnaC mutants are considered to be defective in DNA initiation. Some of the data are consistent with the view that the dna-325 mutation is temperature sensitive in the process of DNA initiation rather than DNA chain elongation: (1) more than two cell divisions occur after a shift to 40 degrees C; (2) upon a shift down to 30 degrees C, cell division occurs again only after the DNA content of the cells has doubled; (3) 80% more DNA is made at 30 degrees C in the presence of chloramphenicol after prior inhibition of DNA synthesis at 40 degrees C. These three observations indicate that rounds of DNA replication were completed at 40 degrees C. Also (4) infective lambda particles can be made at 40 degrees C long after bacterial DNA replication has ceased. It appears however that some DNA initiation can occur at 40 degrees C since (1) a limited amount of DNA synthesis does occur at 40 degrees C after prior alignment of the chromosomes by amino acid starvation at 30 degrees C, and (2) after incubation in bromouracil at the restrictive temperature, heavy DNA is found with both strands containing bromouracil.     

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.     

Genetic control of DNA initiation in Escherichia coli

We describe the isolation, and properties of a mutant (CT28) of Escherichia coli with a temperature-sensitive defect in DNA initiation that is reversible. The mutation (dna-28) responsible for this defect is shown to be located in the same region of the map as the dnaC group of DNA initiation mutants.A terminalized culture of CT28 initiates DNA synthesis synchronously immediately upon lowering the temperature, and will do so in the presence of chloram-phenicol.During prolonged incubation at the non-permissive temperature, the cells accumulate a capacity to initiate multiple rounds of replication per chromosome.The variation in the susceptibility of the argH^? and thyA^? alleles to reversion by pulse mutagenesis with nitrosoguanidine during a synchronous round of DNA replication, suggests that this round of replication is bidirectional and commences from an origin in the vicinity of 60 to 65 minutes.CT28 contains two temperature-sensitive mutations. These have been mapped and separated into two derivative strains. One of these, CT28-3b, carries the dna-28 mutation of the C locus, and like the parental double mutant is reversibly temperature-sensitive for an initiation function; but it is more temperature-sensitive than either the double mutant or the other single mutant derivative, CT28-1. The other, CT28-1, is not defective in DNA replication or initiation of replication at the non-permissive temperature.     

Growth and reactivation of single stranded DNA phage phiX174 in E. coli undergoing "thymineless death".

Summary Flac maintenance was aberrant at permissive temperature in a temperature-sensitive dnaC mutant of Salmonella typhimurium when the normally resident pLT2 plasmid was present. Flac was, however, efficiently transferred into the dnaC pLT2⁺ strain and the resulting Flac derivative was almost as efficient in transferring Flac as were dnaC ⁺ pLT2⁺ Flac strains indicating that aberrant Flac maintenance was not associated with appreciable inhibition of transfer replication. A range of F-like plasmids behaved like pLT2 in causing aberrant Flac maintenance when present in the dnaC pLT2^- strain. Flac was, however, stably maintained in the dnaC strain in the absence of other plasmids. Although the F-like plasmids destabilized Flac, each was stably maintained when introduced into strain 11G dnaC pLT2⁺ and pLT2 was also apparently stable under these conditions. The destabilizing effect of pLT2 and other fi ⁺ plasmids was not consequent upon their inhibiting the formation of a repressible F transfer component needed for Flac replication in the dnaC strain. Incompatibility between Flac and the other plasmids induced by the dnaC lesion also appeared unlikely to be a cause of the aberrant Flac maintenance. The possibility is discussed that the initiation of Flac replication differs from that of pLT2 and the F-like plasmids with F competing less effectively than the others for the DnaC gene product.     

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.     

A temperature-sensitive Escherichia coli mutant defective in DNA replication: dnaA, a new gene adjacent to the dnA, gene

Summary An Escherichia coli mutant defective in replication of the chromosome has been isolated from temperature-sensitive mutants that cannot support colicin E1 plasmid DNA synthesis in the presence of chloramphenicol. Cellular DNA synthesis of the mutant ceases almost immediately after transfer to the nonpermissive temperature. The defect is due to a single mutation, dna-59, which is located close to the sites of dnaA mutations and a cou ^R mutation conferring DNA gyrase with resistance to coumermycin. The dna-59 mutant is not able to support DNA synthesis of phage at the high temperature. The mutant also restricts growth of X174 phage at the high temperature, but permits formation of supercoiled closedcircular duplex replicative intermediates. T7 phage can grow on the mutant even at the high temperature.A specialized transducing phage imm ²¹[tna dnaA]#2 (Miki et al., 1978) supports growth of dna-59, dnaA46 and dna-167 cells at the high temperature. Some of the EDTA-resistant derivatives of the phage have lost part or all of the dnaA gene, but carry gene function complementing the defect of dna-59 cells, as judged by conversion of the above dna strains to wild type cells by phage infection, and by suppression of the loss of viability of dna-59 cells at the high temperature by phage infection. The gene containing the dna-59 mutation site is thus distinct from the dnaA gene. Since the dna-59 mutation does not affect expression of the cou ^r gene of DNA gyrase, which is another known gene involved in DNA synthesis near the dnaA gene, this mutation is probably in a new gene, dnaN. From analysis of the suppression activities of imm ²¹[tna dnaA]#2 phage and its deletion derivatives against dnaN59 cells, it is suggested that the expression of the dnaN gene function is reduced by deletion in the dnaA region.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

Imparired DNA synthesis and envelope defect in a mutant of Salmonella typhimurium

Summary S. typhimurium mutants with temperature-sensitive synthesis of DNA have been isolated. One of these mutants,dna-26, has been studied in detail. DNA synthesis is stopped indna-26 without any residual replication after shift to 42° though increase in cell mass is not inhibited. Mutantdna-26 shows increased sensitivity to deoxycholate, to nalidixic acid and rifampicin. This suggests a cell envelope defect. Inhibition of DNA synthesis at 42° can be phenotypically cured indna-26 by 0.25 M NaCl and KCl and 0.44 M sucrose but not by 0.44 M glycerol. This DNA synthesis induced by hypertonic medium proceeds at a slower rate than increase in cell mass but is predominantly due to normal sequential chromosome replication. The position of mutationdna-26 has been approximately mapped in thepurD region of the chromosome.     

Temperature sensitive initiation of DNA synthesis in a mutant of Salmonella typhimurium

Summary A mutant (dna-1) of Salmonella typhimurium defective in DNA synthesis is described. DNA synthesis is stopped in this mutant at 42° after a residual synthesis amounting to about 50 to 60% of the total cellular DNA in minimal medium and about 120 to 200% in a medium enriched with amino acids. Reshift back to permissive temperature after the inhibition of DNA synthesis at 42° allows for recovery of DNA synthesis after a lag of about 30 min. Protein synthesis is required during that lag for the recovery of DNA synthesis at permissive temperature. The density transfer experiments indicate that in the mutant dna-1 chromosome termini are replicated normally at 42° while the initiation of new rounds of replication is inhibited although the mutation is probably leaky at this temperature. The mutant is hypersensitive to sodium deoxycholate at 42° which suggests alteration of the membrane structure.     

Complementation of a dnaC initiation defect in vitro

Summary 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.     

The initiation of chromosome replication in a dnaAts46 and a dnaA+ strain at various temperatures

Summary The regulation of chromosome replication initiation was studied at various temperatures with an E. coli dnaA46 strain and its dnaA ⁺ parent. We find that, in both strains, the initiation mass varies depending upon growth temperature while the replication time remains constant relative to the cell doubling time. In the permissive temperature range, the initiation mass of the dnaA46 mutant strain is larger by a constant factor than that for a dnaA ⁺ strain. We conclude that, even at temperatures permissive for growth of the dnaA46 strain, the activity of the dnaA46 product is lower than that of the wild-type protein. The dnaA gene product, therefore, plays an important role in regulating initiation.     

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.     

Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

Summary The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30° C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA ⁺, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42° to 30° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42° C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA ⁺ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.A preliminary account of this work was presented at the Annual Meeting of the Molecular Biology Society of Japan in 1989.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.