Discontinuity in DNA replication during expression of accumulated initiation potential in dnaA mutants of Escherichia coli.

Potential for initiation of chromosome replication present in temperature-sensitive, initiation-defective dnaA5 mutants of Escherichia coli B/r incubated at nonpermissive temperature was expressed by shifting to a more permissive temperature (25 degrees C). Upon expression of initiation potential, the rate of [3H]thymidine incorporation varied in a bimodal fashion, i.e., there was an initial burst of incorporation, which lasted 10 to 20 min, then a sudden decrease in incorporation, and finally a second rapid increase in incorporation. Analyses of this incorporation pattern indicated that a round of replication initiated upon expression of initiation potential, but DNA polymerization stopped after replication of 5 to 10% of the chromosome. This round of replication appeared to resume about 30 min later coincident with initiation of a second round of replication. The second initiation was unusually sensitive to low concentrations of novobiocin (ca. 1 microgram/ml) when this inhibitor was added in the presence of chloramphenicol. In the absence of chloramphenicol, novobiocin at this concentration had no detectable effect on DNA replication. It is suggested that cis-acting inhibition, attributable to an attempted second initiation immediately after the first, caused the first round to stall until both it and the second round could resume simultaneously. This DNA replication inhibition, probably caused by overinitiation, could be a consequence of restraints on replication in the vicinity of oriC, possibly topological in nature, which limit the minimum interinitiation interval in E. coli.     

Suppression of the Escherichia coli dnaA46 mutation by a mutation in trxA, the gene for thioredoxin

Summary The dasC mutation, an extragenic suppressor of dnaA46, was mapped by P1 transduction near the rep, trxA, rho region of the Escherichia coli chromosome. The dasC mutation could not be separated from trxA by P1 transduction indicating that dasC and trxA are allelic. Multicopy plasmids containing an intact trxA gene were able to reverse the suppressive effect of the dasC mutation on the dnaA46 mutation. Introduction of a frameshift mutation into the cloned trxA coding region abolished the ability of these recombinant plasmids to reverse the suppressive effect. These results indicate that dasC is allelic with trxA, the gene encoding thioredoxin.     

Cardiolipin activation of dnaA protein, the initiation protein of replication in Escherichia coli

ATP binding to dnaA protein is essential for its action in initiating the replication of plasmids that bear the unique origin of the Escherichia coli chromosome (oriC). ADP bound to that site renders dnaA protein inactive for replication. Diphosphatidylglycerol (cardiolipin), a diacidic membrane phospholipid, displaces the bound nucleotide, and in the presence of components that reconstitute replication, fully reactivates the inert ADP form of dnaA protein. The monacidic phosphatidylglycerol is one-tenth as active as cardiolipin, whereas the neutral phosphatidylethanolamine, the principal E. coli phospholipid, is inactive. Fluphenazine, a tranquilizer drug, blocks cardiolipin activation of dnaA protein, in keeping with the inhibitory action of such agents on phospholipid-dependent enzymes. With the use of this drug to terminate cardiolipin action, dependence of the activation on time, elevated temperature, and high levels of ATP was demonstrated. Cardiolipin binding of nucleotide-free dnaA protein prevents binding of ATP and initiation of oriC replication. Removal of a fatty acid from cardiolipin by phospholipase A reverses this inhibitory effect. The strong and specific interaction of cardiolipin, a cell membrane component, with an essential nucleotide-binding site of dnaA protein, the protein essential for the initiation of chromosome replication, may be an important element in regulating the cell cycle.     

Control of the initiation of DNA replication in Escherichia coli. II. Function of the dnaA product.

Summary General growth parameters and the kinetics of DNA replication have been determined in merogenotes carrying different combinations of the dnaA⁺ and the dnaA5 allele. The strain which is homozygous diploid for dnaA5 is different from all other combinations in cell volume, DNA per mass ratio, number of replication points per chromosome, and polymerization rate of DNA. From this we deduce that the dnaA product is a positively acting regulatory protein in initiation.In an appendix we show that in combinations between the dnaA5 and dnaA204 alleles the phenotype of dnaA5 is dominant.     

Roles of Escherichia coli histone-like protein HU in DNA replication: HU-beta suppresses the thermosensitivity of dnaA46ts

The HU protein is a small, basic, heat-stable DNA-binding protein that is well-conserved in prokaryotes and is associated with the bacterial nucleoid. In enterobacteria, including Escherichia coli, HU is a heterotypic dimer, HUalphabeta, composed of two closely related sub-units encoded by the hupA and hupB genes, respectively. HU was shown to participate in vitro in the initiation of DNA replication as an accessory factor to assist the action of DnaA protein in the unwinding of oriC DNA. To further elucidate the role of HU in the regulation of the DNA replication initiation process, we tested the synchrony phenotype in the absence of either one or both HU sub-units. The hupAB mutant exhibits an asynchronous initiation, the hupA mutant shows a similar reduced synchrony, whereas the hupB mutant shows a normal phenotype. Using a thermosensitive dnaA46 strain (dnaA46ts), an initiation mutant, we reveal a special role of HUbeta. The presence of a plasmid overproducing HUbeta in a dnaA46ts lacking HU (hupAB background) compensates for the thermosensitivity of this initiation mutant. Moreover, the overproduction of HUbeta confers to dnaA46ts a pattern of asynchrony similar to that of a dnaAcos, the intragenic suppressor of dnaA46ts. We show that the relative ratio of HUalpha versus HUbeta is greatly perturbed in dnaA46ts which accumulates little, if any, HUbeta. Therefore, the suppression of thermosensitivity in dnaA46hupAB by HUbeta may be caused by an unexpected absence of HUbeta in the dnaA46ts mutant. Visibly the HU composition is sensitive to the different states of DnaA, and may play a role during the regulation of the initiation process of the DNA replication by affecting subsequent events along the cell cycle.     

Interaction of dnaA46 protein with a stimulatory protein in replication from the Escherichia coli chromosomal origin

Highly purified preparations of dnaA46 protein have permitted its biochemical characterization in comparison with the activities of wild type dnaA protein. We have determined that dnaA46 protein was reduced in its ability to bind to DNA fragments containing oriC. This mutant protein was also defective in binding ATP and was inactive for replication of oriC-containing plasmids in purified enzyme systems. In contrast, dnaA46 protein was active for oriC plasmid replication when added to reactions containing a crude enzyme fraction deficient in dnaA protein. One or more proteins have been identified which appear to interact with dnaA46 protein prior to DNA synthesis. These studies suggest that this interaction is thermolabile. Stimulation of dnaA46 protein activity resulted in a reduction of the prolonged lag prior to DNA synthesis.     

dnaK protein stimulates a mutant form of dnaA protein in Escherichia coli DNA replication

Two proteins have been identified which stimulate a mutant form of dnaA protein in replication of plasmids containing the chromosomal origin, oriC. One of these is dnaK protein by the criteria of (i) absence of stimulatory activity in enzyme fractions from dnaK mutants, (ii) elevated levels of stimulatory activity in fractions from a dnaK protein overproducer, (iii) comigration of the stimulatory protein with authentic dnaK protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iv) replacement of this stimulatory protein by dnaK protein in stimulation assays. The stimulatory effect of dnaK protein on dnaA46 protein in replication suggests that this interaction, occurring prior to its action in DNA replication, may regulate its activity.     

Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes

Summary A hybrid phage (Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA ⁺-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.     

Increased expression of the dnaA gene has no effect on DNA replication in a dnaA+ strain of Escherichia coli

We have constructed a pBR322 plasmid derivative which expresses dnaA protein under the control of the E. coli lac UV5 promotor. Expression of the dnaA protein from the plasmid is inducible by isopropyl-beta-D-thiogalactoside. In a dnaA+ strain induction has no effect on the accumulation of DNA. In contrast, in a thermosensitive dnaA46 strain, induction, at either the permissive or the nonpermissive temperature, results in an immediate stimulation of DNA accumulation. We conclude that, while in a dnaA46 strain dnaA protein limits DNA replication, in a dnaA+ strain dnaA protein activity does not control the timing of replication initiation.     

The nucleoid protein H-NS facilitates chromosome DNA replication in Escherichia coli dnaA mutants.

Growth inhibition of the dnaA(Cs) mutant, which overinitiates chromosome replication, was shown to be dependent upon the nucleoid protein H-NS. [3H]thymine incorporation experiments indicated that the absence of H-NS inhibited overreplication by the dnaA(Cs) mutant. In addition, the temperature-sensitive phenotype of a dnaA46 mutant was enhanced by disruption of H-NS. These observations suggest that H-NS directly or indirectly facilitates the initiation of chromosome replication.     

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.     

Cooperation of the prs and dnaA gene products for initiation of chromosome replication in Escherichia coli.

A new Escherichia coli mutant allele, named dnaR, that causes thermosensitive initiation of chromosome replication has been identified to be an allele of the prs gene, the gene for phosphoribosylpyrophosphate synthetase (Y. Sakakibara, J. Mol. Biol. 226:979-987, 1992; Y. Sakakibara, J. Mol. Biol. 226:989-996, 1992). The dnaR mutant became temperature resistant by acquisition of a mutation in the dnaA gene that did not affect the intrinsic activity for the initiation of replication. The suppressor mutant was capable of initiating replication from oriC at a high temperature restrictive for the dnaR single mutant. The thermoresistant DNA synthesis was inhibited by the presence of the wild-type dnaA allele at a high but not a low copy number. The synthesis was also inhibited by an elevated dose of a mutant dnaR allele retaining dnaR activity. Therefore, thermoresistant DNA synthesis in the suppressor mutant was dependent on both the dnaA and the dnaR functions. On the basis of these results, I conclude that the initiation of chromosome replication requires cooperation of the prs and dnaA products.     

Suppressor genes of a dnaA temperature sensitive mutation in Escherichia coli

Summary Recombinant plasmids were constructed from EcoRI digests of Escherichia coli chromosomal DNA and pMB9 DNA by selecting for suppression of a dnaA–T46 temperature-sensitive mutation. Two types of plasmid capable of suppressing the dnaA mutation were isolated. They did not carry any genetic markers around dnaA and physical mapping with various restriction enzymes showed that neither of the plasmids contained the dnaA gene. One plasmid, pYT47, was characterized further and the protein responsible for the suppression was identified by two-dimensional gel electrophoresis. The molecular weight of the suppressor protein was about 68 Kdal and thus is clearly different from the dnaA gene product.     

Suppressors of a UGG missense mutation in Escherichia coli

As part of our investigation of tRNA structure-function relationships, we isolated and preliminarily characterized translational suppressors of the tryptophan codon UGG in a trpA missense mutant of Escherichia coli. the parent strain also contained two other mutant alleles relevant to the suppressor search; these were supD, which codes for a serine-inserting amber suppressor tRNA, and gly V55, the gene for a GGA/G-reading mutationally altered glycine tRNA. On the basis of map location, reversed-phase (RPC-5) column chromatography of glycyl-tRNA, and codon response, several classes have been distinguished so far. The number of suppressors in each class, their codon responses, and their apparent genic identities, respectively, are as follows: class 1--4 suppressors, UGG, supD; class 2--12 suppressors, UGG, glyU; class 3--9 suppressors, UGA and UGG, glyT; class 4--2 suppressors, UGG, glyT; class 5--7 suppressors, UGG, gly V55. Besides these, one suppressor retains supD activity, but so far its map location has not been distinguished from that of supD. Another suppressor clearly does not map near supD or any of the glycine tRNA genes mentioned. These last two suppressors may represent novel missense suppressors such as misacylated tRNA's or mutationally altered aminoacyl-tRNA synthetases, tRNA modification enzymes, or ribosomes. Finally, three other suppressors were obtained from a strain containing glyT56, the gene for an AGA/G-reading form of glyT tRNA. All three occurred at the expense of glyT56 activity and exhibited the the transductional linkage to argH that is characteristic of glyT.     

Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12.

The product of the dnaA gene is essential for the initiation of chromosomal DNA replication in Escherichia coli K-12. A cold-sensitive mutation, dnaA(Cs), was originally isolated as a putative intragenic suppressor of the temperature sensitivity of a dnaA46 mutant (G. Kellenberger-Gujer, A. J. Podhajska, and L. Caro, Mol. Gen. Genet. 162:9-16, 1978). The cold sensitivity of the dnaA(Cs) mutant was attributed to a loss of replication control resulting in overinitiation of DNA replication. We cloned and sequenced the dnaA gene from the dnaA(Cs) mutant and showed that it contains three point mutations in addition to the original dnaA46(Ts) mutation. The dnaA(Cs) mutation was dominant to the wild-type allele. Overproduction of the DnaA(Cs) protein blocked cell growth. In contrast, overproduction of wild-type DnaA protein reduced the growth rate of cells but did not stop cell growth. Thus, the effect of elevated levels of the DnaA(Cs) protein was quite different from that of the wild-type protein under the same conditions.