E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration

The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.     

Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome. The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact and functional oriC sequence. The seqA2 mutation was found to overcome the incompatability phenotype by increasing the cellular oriC copy number 3-fold thereby allowing minichromosomes to coexist with the chromosome. The replication pattern of a wild-type strain with multiple integrated minichromosomes in the oriC region of the chromosome, led to the conclusion that initiation of DNA replication commences at a fixed cell mass, irrespective of the number of origins contained on the chromosome.     

Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.     

Interviewers'' perceptions of persons-organization fit and organizational selection decisions

The Escherichia coli DnaA protein is a sequence-specific DNA binding protein that promotes the initiation of replication of the bacterial chromosome, and of several plasmids including pSC101. Twenty-eight novel missense mutations of the E. coli dnaA gene were isolated by selecting for their inability to replicate a derivative of pSC101 when contained in a lambda vector. Characterization of these as well as seven novel nonsense mutations and one in-frame deletion mutation are described here. Results suggest that E. coli DnaA protein contains four functional domains. Mutations that affect residues in the P-loop or Walker A motif thought to be involved in ATP binding identify one domain. The second domain maps to a region near the C terminus and is involved in DNA binding. The function of the third domain that maps near the N terminus is unknown but may be involved in the ability of DnaA protein to oligomerize. Two alleles encoding different truncated gene products retained the ability to promote replication from the pSC101 origin but not oriC, identifying a fourth domain dispensable for replication of pSC101 but essential for replication from the bacterial chromosomal origin, oriC.     

Expression of zonula occludens and adherens junctional proteins in human venous and arterial endothelial cells: role of occludin in endothelial solute barriers

ColE1-derived plasmids containing different recombinant genes which are controlled by the tac promoter were amplified following induction with IPTG, but no amplification occurred if product formation was not induced. The plasmid copy number of recombinant E. coli increased three- to sixfold within a period of about 6 h in shake flask experiments, batch cultures, and glucose-limited fed-batch cultivations. Plasmid amplification occurred in E. coli B strains as well as in K-12 strains with different plasmids (rop+ and rop-) coding for various heterologous proteins. The amplification was not caused by a toxic effect of IPTG, but was related to a strong inhibition of translation and chromosomal replication after the induction of heterologous gene expression. Similar to the amplification after chloramphenicol addition, plasmid replication proceeded even if oriC replication and translation were inhibited following strong induction of a recombinant gene. In accordance with the effect of chloramphenicol, the level of ppGpp, which is a negative regulator of ColE1 derived plasmid replication, decreased after induction.     

The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin

The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.     

Primase couples leading- and lagging-strand DNA synthesis from oriC

Coupling of leading- and lagging-strand DNA synthesis at replication forks formed at Escherichia coli oriC has been studied in vitro using a replication system reconstituted with purified proteins. At low concentrations of primase (8 nM), the major replication products were multigenome-length molecules, generated by a rolling circle-type mechanism, and unit-length molecules. Rolling circle DNA replication was inhibited at high concentrations of primase (80 nM) and the major replication products were half-unit-length leading strands and a distinct population of short Okazaki fragments. At low primase concentrations, an asymmetric mode of DNA synthesis occurred. Each strand was made independently and initiation could occur outside of oriC. At high primase concentrations, initiation occurred exclusively at oriC and two coupled replication forks proceeded bidirectionally around the plasmid. Presumably, at low concentrations of primase, DnaB (the replication fork helicase) unwound the plasmid DNA before replication forks could form, leading to initiation at sites other than oriC. On the other hand, high concentrations of primase resulted in successful capture of the helicase leading to the formation at oriC of coupled replication forks capable of coordinated leading- and lagging-strand synthesis.     

Insulin resistance is a common denominator of post-transplant diabetes mellitus and impaired glucose tolerance in renal transplant recipients

The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5'-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158.     

In vivo evidence for the involvement of anionic phospholipids in initiation of DNA replication in Escherichia coli

In vitro, anionic phospholipids can reactivate inactivated DnaA protein, which is essential for initiation of DNA replication at the oriC site of Escherichia coli [Sekimizu, K. & Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135]. Mutations in the pgsA gene (encoding phosphatidylglycerophosphate synthase) limit the synthesis of the major anionic phospholipids and lead to arrest of cell growth. We report herein that a mutation in the rnhA gene (encoding RNase H) that bypasses the need for the DnaA protein through induction of constitutive stable DNA replication [Kogoma, T. & von Meyenburg, K. (1983) EMBO J. 2, 463-468] also suppressed the growth arrest phenotype of a pgsA mutant. The maintenance of plasmids dependent on an oriC site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein.     

Characterization of the oriC region of Mycobacterium smegmatis

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.     

Tattooing increases the number of Langerhans cells in skin: an immunocytochemical study

A pTSK series of recombinant plasmids were constructed by cloning DNA fragments of pXZ10145 or its deleted deriviate pATN65 into plasmid vector pACYC177 of E. coli. Experiment results of Coryneform bacteria transformation with these pTSK plasmids allowed us to localize the essential region for self-replication on plasmid pXZ10145. The minimal replication region of the pXZ10145 was located on a 1.2kb Nael-Nrul DNA fragment in which only one open reading frame was found. This ORF was believed to be encoded a trans-acting replication factor. The replication origin (oriV) was locate on a 0.3kb NaeI-SalI fragment which was within the ORF region.     

The ribosomal S16 protein of Escherichia coli displaying a DNA-nicking activity binds to cruciform DNA

We have recently shown that the ribosomal S16 protein of Escherichia coli is a magnesium-dependent DNase which introduces nicks into supercoiled DNA molecules [Oberto, J., Bonnefoy, E., Mouray, E., Pellegrini, O., Wikstrom, P. M. & Rouvière-Yaniv, J. (1996) Mol. Microbiol. 19, 1319-1330]. In this work we analysed the DNA-binding and DNA-nicking properties of S16 using two different approaches. Gel-retardation assays showed that S16 is a structure-specific DNA-binding protein displaying a preferential binding for cruciform DNA structures. This specific binding to cruciform DNA was further investigated using a supercoiled plasmid carrying the origin of replication of E. coli (oriC) which is an (A+T)-rich DNA region with abundant palindromic sequences susceptible of forming cruciform-like structures in vivo. We show that the nicks introduced by S16 in oriC are not randomly positioned but are precisely localised near such palindromic sequences. In addition, the nicking activity of S16 appeared to be sequence dependent since the cuts introduced by S16 occurred next to an adenine, in most cases an unpaired adenine, usually followed by a GTT sequence. Overall these experiments indicate that S16 requires a cruciform-like DNA structure to bind DNA and the presence of a particular sequence in order to introduce specific single-stranded cuts into a DNA molecule.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.     

Effect on DNA topology by DnaA protein, the initiation factor of chromosomal DNA replication in Escherichia coli

We examined effects on supercoiled DNA topology of DnaA protein, the initiator protein of chromosomal DNA replication in Escherichia coli. The activity was identified in an analysis of plasmid DNA incubated with DnaA protein and DNA topoisomerase I. In Superose 12 gel filtration chromatography, the activity coeluted with DnaA protein. Incubation of DnaA protein with DNA at temperatures over 24 degrees C was required for this activity, which was observed with either oriC plasmid or the replicative form I of phi X174 with no DnaA box. As binding of ATP or ADP to DnaA protein prevented the activity of DnaA protein on DNA topology, binding of the adenine nucleotide may regulate the activity.     

Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups

Two hundred ninety-seven bacteria carrying plasmids that range in size from 5 to 250 kb were identified from more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments. While some isolates contained numerous (three to five) small (5- to 10-kb) plasmids, the majority of the natural isolates typically contained one large (40- to 100-kb) plasmid. By the method of plasmid isolation used in this study, the frequency of plasmid incidence ranged from 24 to 28% depending on the samples examined. Diversity of the plasmids occurring in the marine sediment bacterial populations was examined at the molecular level by hybridization with 14 different DNA probes specific for the incompatibility and replication (inc/rep) regions of a number of well-characterized plasmid incompatibility groups (repB/O, FIA, FII, FIB, HI1, HI2, I1, L/M, X, N, P, Q, W, and U). Interestingly, we found no DNA homology between the plasmids isolated from the culturable bacterial population of marine sediments and the replicon probes specific for numerous incompatibility groups developed by Couturier et al. (M. F. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Our findings suggest that plasmids in marine sediment microbial communities contain novel, as-yet-uncharacterized, incompatibility and replication regions and that the present replicon typing system, based primarily on plasmids derived from clinical isolates, may not be representative of the plasmid diversity occurring in some marine environments. Since the vast majority of marine bacteria are not culturable under laboratory conditions, we also screened microbial community DNA for the presence of broad- and narrow-host-range plasmid replication sequences. Although the replication origin of the conjugally promiscuous broad-host-range plasmid RK2 (incP) was not detectable in any of the plasmid-containing culturable marine isolates, DNA extracted from the microbial community and amplified by PCR yielded a positive signal for RK2 oriV replication sequences. The strength of the signal suggests the presence of a low level of the incP replicon within the marine microbial community. In contrast, replication sequences specific for the narrow-host-range plasmid F were not detectable in DNA extracted from marine sediment microbial communities. With the possible exception of mercuric chloride, phenotypic analysis of the 297 plasmid-bearing isolates did not demonstrate a correlation between plasmid content and antibiotic or heavy metal resistance traits.     

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro

It was demonstrated previously that replication of plasmids derived from bacteriophage lambda (so-called lambda plasmids) is inhibited in wild-type Escherichia coli cells starved for isoleucine and arginine whereas it proceeds under the same conditions in relA mutants. Since replication of other replicons during the stringent or relaxed response depends on the nature of the deprived amino acid, we investigated replication of lambda plasmids in E. coli relA+ and relA- strains starved for different amino acids. We found that replication of lambda plasmids is generally inhibited during the stringent, but not relaxed, response. Differences between cells starved for different amino acids, although reproducible, were not dramatic. Amino acid starvation was previously proposed as a method for amplification of lambda plasmid DNA in vivo. We found that during amino acid limitation lambda plasmids replicate more extensively in the relA mutants than during amino acid starvation. The efficiency of plasmid DNA amplification was found to be dependent on the kind of limited amino acid; in relA- bacteria limited for leucine we observed about 10-fold plasmid amplification. Some lambda plasmid replication was also found under these conditions in the relA+ host. The mechanism of the stringent control of lambda plasmid DNA replication has already been proposed. Here the possible mechanism of the regulation of lambda plasmid replication during amino acid limitation is presented.     

An ultrasonic system for diameter pulse tracking in arteries: problems and pitfalls

DnaA protein and the Escherichia coli chromosomal origin (oriC) form an initial complex at an early stage in the initiation of DNA replication. We have used electron microscopy to determine which structure among the several formed in the reconstitution of this multicomponent system is the replicatively active complex. One distinctive structure could be correlated with activity and localized to oriC, whilst several others could not. Formation of an open complex in the next stage of initiation was accompanied by the presence of a structure similar in size and shape to that of the functional initial complex. Whereas the initial complex was observed with either ATP or the ADP-forms of DnaA protein, only the ATP-form was effective in producing the open complex. Mutagenesis of several DNA sequence elements in oriC, known to be important for replication, was employed to determine the effects of these alterations on formation of the initial complex. As judged by electron microscopy and by functional assays, the region containing the four 9-mer dnaA boxes proved to be essential for the formation of the initial complex, while the three contiguous AT-rich 13-mers, known sites for opening of oriC, were not.     

Site-directed mutational analysis for the ATP binding of DnaA protein. Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.