The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication.

Examination of the effect of the himA and himD mutants of E. coli on the maintenance of plasmid R6K has revealed that the gamma origin-containing replicons cannot be established in any of the mutants deficient in the production of E. coli Integration Host Factor (IHF). Contrary, the R6K derivatives containing other origins of the plasmid (alpha and/or beta) replicate in a host lacking functional IHF protein. We show that IHF protein binds specifically to a segment of the replication region which is essential for the activity of all three R6K origins. Mapping the IHF binding sequence with neocarzinostatin showed that the protein protects three segments of the origin: two strong binding sites reside within an AT-rich block, while the third, considerably weaker site is separated from the other two by a cluster of the seven 22 bp direct repeats. These seven repeats have been shown previously to bind the R6K-encoded initiator protein pi. We also demonstrate that the establishment of pi-origin complexes prior to IHF addition prevents the binding of the IHF protein to the gamma origin. The binding sequences of IHF and pi proteins do not overlap, therefore, we propose that the binding of pi protein alters the structure of the DNA and thereby prevents the subsequent binding of IHF protein.     

The replication of plasmid pSC101

The origin of replication of plasmid pSC101 presents features reminiscent of those found in a number of plasmids. As for those plasmids, many details about the way it initiates its replication are beginning to be known, but the regulation of this process will not be easily understood.     

Maintenance of plasmid pSC101 in Escherichia coli requires the host primase.

The abilities of three Escherichia coli strains with thermosensitive dnaG alleles to maintain plasmids pSC101 or pBR322 or an RP4 derivative were studied at elevated growth temperatures. Under these conditions, pSC101 segregated from cells to a greater extent than did pBR322. No segregation of the primase-encoding RP4 derivative was observed.     

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.     

The pSC101 par locus alters protein-DNA interactions in vivo at the plasmid replication origin.

We report here direct evidence that mutations in the par locus affect protein-DNA interactions in vivo at the replication origin of plasmid pSC101. Concomitant with par-mediated plasmid stabilization, two sites in the origin region show an altered methylation pattern as detected by in vivo footprinting with dimethyl sulfate. One site is located near an integration host factor-binding sequence adjacent to the first of three direct repeats known to be involved in the initiation of pSC101 replication; the second site is within the third direct repeat.     

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.     

Effects of the pSC101 partition (par) locus on in vivo DNA supercoiling near the plasmid replication origin.

Previous work has shown that deletion of the partition (par) locus of plasmid pSC101 results in decreased overall superhelical density of plasmid DNA and concommitant inability of the plasmid to be stably inherited in populations of dividing cells. We report here that the biological effects of par correlate specifically with its ability to generate supercoils in vivo near the origin of pSC101 DNA replication. Using OsO4 reactivity of nucleotides adjoining 20 bp (G-C) tracts introduced into pSC101 DNA to measure local DNA supercoiling, we found that the wild type par locus generates supercoiling near the plasmid's replication origin adequate to convert a (G-C) tract in the region to Z form DNA. A 4 bp deletion that decreases par function, but produces no change in the overall superhelicity of pSC101 DNA as determined by chloroquine/agarose gel analysis, nevertheless reduced (G-C) tract supercoiling sufficiently to eliminate OsO4 reactivity. Mutation of the bacterial topA gene, which results in stabilized inheritance of par-deleted plasmids, restored supercoiling of (G-C) tracts in these plasmids and increased OsO4 reactivity in par+ replicons. Removal of par to a site more distant from the origin decreased supercoiling in a (G-C) tract adjacent to the origin and diminished par function. Collectively, these findings indicate that par activity is dependent on its ability to produce supercoiling at the replication origin rather than on the overall superhelical density of the plasmid DNA.     

A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K.

Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.     

Autogenous regulation of synthesis of the replication protein in plasmid pSC101

Summary A 1.3-kb segment of plasmid pSC101 includes the replication origin (ori) and the gene (rep) encoding the 37 kilodalton (K) protein required for autonomous replication of the plasmid. The present work describes the regulation of the rep gene expression. The promoters P _R and P _L fail to promote rep gene expression when located upstream of a sequence with dyad symmetry overlapping the rep promoter, whereas elimination of this sequence allows expression and results in over-production of the rep protein. When expression of trpA-lacZ is controlled under the rep promoter, -galactosidase is produced without the lac inducer. However, this enzyme synthesis is effectively reduced when the complete rep sequence is provided in trans. A partial disruption of the sequence with dyad symmetry relieves the repression. These results suggest that expression of the rep gene is negatively regulated by its own product and that the sequence with dyad symmetry plays the role of a receptor site for the rep protein.Abbreviations bp base pairs - kb kilobase pairs - ORF open reading frame - SDS sodium dodecyl sulphate - ts temperature sensitive     

Minimal essential origin of plasmid pSC101 replication: requirement of a region downstream of iterons.

The minimal replication origin (ori) of the plasmid pSC101 was defined as an about 220-bp region under the condition that the Rep (or RepA) protein, a plasmid-encoded initiator protein, was supplied in trans. The DnaA box is located at one end of ori, as in other plasmids, like mini-F and P1. The other border is a strong binding site (IR-1) of Rep which is palindromic sequence and lies in an about 50-bp region beyond the repeated sequences (iterons) in ori. This IR-1 is located just upstream of another strong Rep binding site (IR-2), the operator site of the structure gene of Rep (rep), but its function has not been determined. The present study shows that the IR-1 sequence capable of binding to Rep is essential for plasmid replication with a nearly normal copy number. Furthermore, a region between the third iteron and IR-1 is also required in a sequence-specific fashion, since some one-base substitution in this region inactivate the origin function. It is likely that the region also is a recognition site of an unknown protein. Three copy number mutations of rep can suppress any one-base substitution mutation. On the other hand, the sequence of a spacer region between the second and the third iterons, which is similar to that of the downstream region of the third iteron, can be changed without loss of the origin function. The requirement of the region downstream of iterons in pSC101 seems to be unique among iteron-driven plasmid replicons.     

Role of DNA superhelicity in partitioning of the pSC101 plasmid

Previous work has shown that a cis-acting locus (termed par for partitioning) on the pSC101 plasmid accomplishes its stable inheritance in dividing cell populations. We report here that the DNA of pSC101 derivatives lacking the par region shows a decrease in overall superhelical density as compared with DNA of wild-type pSC101. Chemicals and bacterial mutations that reduce negative DNA supercoiling increase the rate of loss of par plasmids and convert normally stable plasmids that have minimal par region deletions into unstable replicons. topA gene mutations, which increase negative DNA supercoiling, reverse the instability of partition-defective plasmids that utilize the pSC101, p15A, F, or oriC replication systems. Our observations show that the extent of negative supercoiling of plasmid DNA has major effects on the plasmid's inheritance and suggest a mechanism by which the pSC101 par region may exert its stabilizing effects.     

The P1 plasmid partition complex at parS. The influence of Escherichia coli integration host factor and of substrate topology

The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.     

Replication of plasmid pSC101 in Escherichia coli K12: requirement for dnaA function.

Summary Protoplasts of soybean and N. glauca were induced to fuse with polyethylene glycol (PEG 1540). Up to 39% of the protoplasts in the treated population were heterokaryocytes. When the heterokaryocytes were isolated and individually cultivated they divided indefinitely and each produced many millions of cells within 2–3 months.The chromosomal behaviour of soybean and N. glauca in the hybrids were not synchronous in the first few cell generations and the chromosomes of N. glauca had a tendency to stick together and break into pieces. However, some of the N. glauca chromosomes were still retained in the somatic hybrids after 6 months of culturing. The chromosomes of the N. glauca were reconstructed in such a way that in the later cell generations, the movement of the N. glauca chromosomes were in synchrony with the soybean chromosomes.NRCC NO. 15668     

The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli

After binding to its four 9-mer boxes in the 245-base pair Escherichia coli replication origin (oriC), dnaA protein effects the formation of an "open complex" in an adjacent region made up of three 13-mers (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). This open complex formation requires the ATP form of dnaA protein assisted by HU protein (Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50, 259-265). We now provide direct evidence that dnaA protein binds the 13-mers, sequences that bear no resemblance to the 9-mer box. The evidence is (i) displacement of dnaA protein from the open complex by oriC or by a synthetic oligonucleotide containing the 13-mers, but not by a mutant of oriC lacking the 13-mers; (ii) filter binding of the synthetic (13-mer) oligonucleotide by dnaA protein; and (iii) requirement for the ATP form of dnaA protein assisted by HU protein for temperature-dependent binding to the 13-mer region. Controlled proteolysis of dnaA protein results in a prompt loss of oriC binding; an NH2-terminal 30-kDa peptide contains the domain that binds ATP and phospholipids known to destabilize the tightly bound ATP.