Suppressor mutations (rin) that specifically suppress the recA+ dependence of stable DNA replication in Escherichia coli K-12

Summary The sdrA102 mutation confers upon cells the ability to replicate DNA in the absence of protein synthesis. This mutation was combined with the recA200 mutation, which renders the recA protein thermolabile, and had little effect on normal replication. However, the sdrA102 recA200 double mutant exhibited temperature-sensitive stable DNA replication: it replicated DNA continuously in the presence of chloramphenicol at 30°C, whereas at 42°C DNA replication ceased after the DNA content increased only 40–45%. Suppressor mutants (rin; recA-independent) capable of stable DNA replication at 42°C were isolated from the double mutant. The suppressor mutant retained all other recA ^– characteristics, i.e., deficient general recombination, severe UV-sensitivity, and incapability of prophage induction in lysogens. This indicates that the rin mutation specifically suppresses the recA ⁺ dependency of stable DNA replication. It is suggested that the recA ⁺ protein stabilizes a specific structure, similar to an intermediate in recombination, which may function in the initiation of stable DNA replication.     

Escherichia coli mutants temperature-sensitive for DNA synthesis

Summary A series of mutants of E. coli temperature-sensitive for DNA synthesis has been studied. The temperature-sensitive DNA mutations map in seven distinct genetic loci most of which have not been previously reported. Mutations in dnaA and in dnaC affect the initiation of DNA replication; those at the remaining loci affect chain elongation. A temperature-sensitive Flac is shown to suppress a group A mutant with somewhat less efficiency than other F factors previously reported by others. The gene products rendered temperaturesensitive by the mutations have not been identified for any of the loci.     

Phenotypes ofdnaA mutants ofEscherichia coli sensitive to phenothiazine derivatives

The activation of DnaA protein by cardiolipin is inhibited by fluphenazinein vitro. We therefore examined the sensitivity of temperature-sensitivednaA mutants ofEscherichia coli to fluphenazine and other phenothiazine derivatives. Among the eightdnaA mutants tested,dnaA5, dnaA46 dnaA602, anddnaA604, mutants with mutations in the putative ATP binding site of DnaA protein, showed higher sensitivities to phenothiazine derivatives than did the wild-type strain. ThednaA508 anddnaA167 mutants, which have mutations in the N-terminal region of DnaA protein, also showed higher sensitivities to phenothiazine derivatives. On the other hand, thednaA204 anddnaA205 mutants, with lesions in the C-terminal region of the DnaA protein, showed the same sensitivity to phenothiazine derivatives as the wild-type strain. Complementation analysis with a plasmid containing the wild-typednaA gene and phage P1-mediated transduction confirmed thatdnaA mutations are responsible for these sensitivity phenotypes.     

RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication

Summary Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh ^- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh ^- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh ^- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh ^- mutations suppressed the temperature-sensitive growth character of dnaA ^ts mutant, (5) rnh ^- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and-independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.Abbreviations AP^r ampicillin-resistant - kb kilobase pair(s) - NEM N-ethyl maleimide - Ts temperature-sensitive     

Regulation of DNA synthesis and capacity for initiation in DNA temperature sensitive mutants of Escherichia coli

Summary The capacity for initiation and subsequent chain elongation was examined in several DNA temperature sensitive mutants of Escherichia coli after the mutants had been held at nonpermissive temperature for approximately 1.5 generation equivalents and then returned to permissive temperature in the presence of chloramphenicol. The results obtained indicate that 4–5 sets of replication forks can be initiated after return to permissive temperature in the presence of chloramphenicol but the forks apparently become stalled and fail to complete chromosomal replication in the presence of chloramphenicol. In temperature reversible dnaA mutants, once the chloramphenicol is removed the forks appear to be able to resume replication at the nonpermissive temperature. The relationship between premature initiation and premature chain termination is discussed.     

The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12

The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism.     

Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli

Summary Extragenic suppressor mutations for dnaA(Ts) mutations mapping in the rpoB gene (-subunit of RNA polymerase) were isolated by selection of spontaneous rifampicin resistant mutants and screening for temperature resistance. Six rpoB mutations were analysed for suppression of 12 different dnaA(Ts) mutations. The analysis showed that all dnaA(Ts) mutations could be suppressed by some rpoB mutation. All six rpoB mutations showed allele specificity when tested for suppression of 12 dnaA (Ts) mutant strains. The allele specificity was found to correlate with the map position of the dnaA (Ts) alleles.     

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinusdnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them. Received: 20 July 1997 / Accepted: 7 October 1997     

DNA lesions that block DNA replication are responsible for the dnaA induction caused by DNA damage

Summary The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 by consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC.     

Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in Escherichia coli is a DnaA-dependent process

Summary The thermosensitivity of dnaA(Ts) mutations can be suppressed by integration of plasmid F (integrative suppression). In the light of the recent finding that F requires DnaA protein for both establishment and maintenance, integrative suppression of 11 dnaA(Ts) mutations by a mini-F, pML31, integrated near oriC was examined. The plating efficiency of integratively suppressed strains was dnaA(Ts) allele-dependent and medium-dependent. The initiation capability of suppressed dnaA(Ts) strains lacking the oriC site and their F^- counterparts was determined at various temperatures between 30°C and 42°C. The degree of integrative suppression measured by the initiation capability varied in a dnaA(Ts) allele-dependent manner. F-directed DNA replication was most affected by the dnaA(Ts) mutations mapping in the middle of the gene whereas oriC-dependent replication was most thermosensitive in strains carrying mutations mapping in the carboxy-terminal half of the gene. The results indicated that the integrative suppression by F plasmid is a DnaA-dependent process and suggested that the requirements for DnaA protein in the oriC-dependent replication and F replication processes are qualitatively different.     

Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli

Summary Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations. The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene. The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene. Several of the mutant dnaA genes were found to have two mutations. The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution. The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA211 each had only one. The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations. Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains. Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein.     

Study on the cell cycle of Bacillus subtilis using temperature-sensitive mutants

Summary Among temperature-sensitive mutants which were defective in septum formation and formed nonseptate filaments at nonpermissive temperatures three (ts31, ts341, ts526) were identified among 434 temperature-sensitive mutants isolated at random from a mutagenized population of Bacillus subtilis 168. The results of morphological observations and characterization of these mutants showed that ts31 and ts341 were septum-initiation mutants and that ts526 was a DNA elongation mutant. The above mutations, and other mutations affecting septum initiation (div355 and tms12) were mapped by PBS1-mediated transduction on the chromosome in three separate regions as follows: pur A16-ts526-div355-cysA14; metC3-(ts31, tms12)-pyrD1-recA1; ebr-2-ts341-uvrA1-hisA1-cysB3. Our results suggest that the initiation process of septum formation requires at least four kinds of gene product. In addition, the sesult obtained with ts526 suggests an intimate connection between septum initiation and DNA replication.     

Requirement of the Escherichia coli dnaA gene function for integrative suppression of dnaA mutations by plasmid R100-1

Summary The phenotype of Escherichia coli dnaA missense and nonsense mutations was integratively suppressed by plasmid R100-1. The suppressed strains, however, could not survive when the dnaA function was totally inactivated. This was demonstrated by the inability of replacing the dnaA allele in the suppressed strain by a dnaA::Tn10 insertion using phage P1-mediated transduction. When the intact dnaA ⁺ allele was additionally supplied by a specialized transducing phage, imm ²¹ dnaA ⁺, which integrated at the att site on the E. coli chromosome, then the dnaA::Tn10 insertion, together with a oriC deletion, were able to be introduced into the suppressed strain. Thus, the mechanisms of dnaA function for oriC and for the replication origin of R100-1 may not be quite the same.     

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.     

Requirement of DNA Gyrase for the initiation of chromosome replication in Escherichia coli K-12

Summary It has been found that strains carrying mutations in the dnaA gene are unusually sensitive to COU, NAL or NOV, which are known to inhibit DNA gyrase activities. The delay in the initiation of chromosome replication after COU treatment has been observed in cells with chromosomes synchronized by amino acid starvation or by temperature shift-up (dnaA46). The unusual sensitivity of growth to COU of the initiation mutant runs parallel to a higher sensitivity to the drug of the initiation of chromosome replication.The double mutant, dnaA46 cou-110 has been isolated and mutation cou-110 conferring resistance of growth, initiation and elongation of chromosome replication to COU was mapped in the gene coding for the subunit of DNA gyrase. The reduced frequency of appearance of the mutants resistant to COU, NAL or NOV in the initiation mutant suggests that some mutations in genes coding for DNA gyrase subunits cannot coexist with the dnaA46 mutation. The possible mechanisms of the requirement of DNA gyrase for dnaA-dependent initiation of E. coli chromosome are discussed.Abbreviations used COU coumermycin A₁ - NAL nalidixic acid - NOV novobiocin     

Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12

Summary Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (oriC) and transposon-insertional inactivation of an initiator gene (dnaA:Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein intornh ^– dnaA: Tn10 and rnh ^– oriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm^–) of the rnh ^– dnaA: Tn10 recA200 strain was suppressed by the presenceof plasmids (pBR322 derivatives) carrying dnaA ⁺only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and pasn20) in the absence of required DnaA⁺ protein nor inhibited dnaA ⁺–dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.     

Maintenance of plasmids in HU and 1HF mutants of Escherichia coli

Summary Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the hupA-hupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the hupA-hupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.     

Inhibition of initiation of mini-F plasmid replication in temperature-sensitive mafA mutants of Escherichia coli K-12

When temperature-sensitive mafA mutants of Escherichia coli K-12 carrying mini-F plasmid (pSC138) are transferred from 30 to 42 °C, plasmid DNA replication as determined by incorporation of [³H]thymidine into covalently closed circular (CCC) mini-F DNA or by DNA-DNA hybridization is inhibited markedly within 10 min. The results of extensive pulse-chase experiments suggest that the initiation rather than the chain elongation step of plasmid replication is affected under these conditions. The replication inhibition in the mutant is accompanied by appearance of a class of plasmid DNA with a buoyant density higher than that of CCC DNA observed in the wild type, and is followed by gradual inhibition of host cell growth. The inhibition of plasmid replication is reversible at least for 60 min under the conditions used, and the recovery at low temperature (30 °C) depends on the synthesis of untranslated RNA. These results taken together with other evidence suggest that the mafA mutations primarily affect the initial step(s) of F DNA replication, presumably at or before the synthesis of untranslated RNA.