Mutational analysis of primosome assembly sites. Evidence for alternative DNA structures

Primosome assembly sites are complex DNA structures that share common functions (they elicit the DNA-dependent ATPase of replication factor Y from Escherichia coli and serve as origins of complementary strand DNA synthesis), but display little sequence homology. In order to ascertain a common basis for factor Y-DNA recognition, a primosome assembly site and its mutated derivatives have been functionally and structurally analyzed. Under conditions in which they lose the capacity to function as ATPase effectors these DNA templates have been (i) assayed for their ability to bind factor Y, and (ii) probed, with pancreatic DNase, for structural alterations. In this ATPase-inactivating environment (suboptimal concentrations of MgCl2 and NaCl, and high levels of the E. coli single-stranded DNA binding protein), factor Y does not bind to its cognate DNA and the DNase cleavage pattern characteristic of this site is perceptibly changed: compared to the DNase digest obtained under activating conditions, cleavage is notably decreased in the 5' half of the site and enhanced at the 3' end. The results of this study strongly indicate that the structure of the primosome assembly site under analysis consists of two hairpins which interact with each other. When the sites of pancreatic DNase attack are plotted on the proposed double hairpin structure, the 5' cleavage sites all map to one duplex while the 3' sites map to the other. The observation that, under factor Y ATPase-activating conditions, the 3' hairpin is largely refractory to the action of pancreatic DNase indicates that tertiary interactions between the two duplexes render a portion of the DNA structure inaccessible to the nuclease.     

Escherichia coli topoisomerase I can segregate replicating pBR322 daughter DNA molecules in vitro

The reconstituted pBR322 DNA replication system has been used to identify a mechanism for the processing and segregation of daughter DNA molecules by Escherichia coli topoisomerase I (Topo I) during the terminal stages of DNA replication. At low concentrations of Topo I (sufficient to confer specificity to the replication system for DNA templates containing a ColE1-type origin of DNA replication), the major products of the replication reaction were: multigenome-length, linear, double-stranded DNA molecules (an aberrant product); multiply interlinked, catenated, supercoiled DNA dimers; and a last Cairns-type replication intermediate. Thirty- to fifty-fold higher concentrations of Topo I led to the appearance of form II and form I pBR322 DNA as the only synthetic products. A model was developed in which Topo I, bound to a single-stranded gap on the parental H strand DNA just upstream of the origin of DNA replication, catalyzed the decatenation of the intermolecular linkages between the two daughter DNA molecules that were generated by primosome-catalyzed unwinding of the residual nonreplicated parental duplex DNA in the last Cairns-type intermediate. At low concentrations of Topo I, however, the intermolecular linkages persisted and, within the context of this replication system, were not removed by DNA gyrase. In support of this model it was demonstrated that: there was a single-stranded gap between the nonreplicated parental duplex region and the 5' end of the nascent leading-strand DNA; the number of intermolecular linkages in the catenated supercoiled DNA dimers was inversely related to the concentration of Topo I; the supercoiled DNA dimers did not serve as a precursor of the final form I DNA product; and maturation of the last Cairns-type replication intermediate to form I DNA was not affected by the presence of coumermycin, a potent inhibitor of the activities of DNA gyrase.     

Mutational analysis of primosome assembly sites. II. Role of secondary structure in the formation of active sites

Based on their activity as effectors for the ATPase activity of Escherichia coli replication factor Y and as templates for primosome-directed DNA synthesis, single-point mutations in the L- and H-strand primosome assembly sites from pBR322 DNA have been grouped into four classes (Abarzúa, P., Soeller, W., and Marians, K. (1984) J. Biol. Chem. 259, 14286-14292). In this report, the effect of various ligands on the characteristic activities of primosome assembly site class II mutants has been examined. Both Mn2+ and spermidine can, at low levels, substitute for Mg2+ in the activation of wild-type sites as effectors for factor Y-catalyzed hydrolysis of ATP. Class II mutant sites characteristically require higher levels of these ligands for activation, suggesting that the specific higher order structure of an active primosome assembly site is maintained through base pairing within the single-stranded DNA sequence. This conclusion is supported by the following. 1) Excess levels of the E. coli single-stranded DNA-binding protein can inactivate wild-type sites at 1 mM Mg2+. Either the addition of NaCl to 80 mM or an increase in the Mg2+ concentration to 5 mM protects against this inactivation. Class II mutant sites, however, cannot be stabilized by 80 mM NaCl at 1 mM Mg2+, and only some class II mutants can be stabilized at 5 mM Mg2+. 2) Active second-site revertants, isolated in vivo and in vitro, of inactive primosome assembly sites containing multiple-base substitutions have mutated to restore lost base pairs in the proposed stem and loop structure of the sites.     

Preferential site-specific hemimethylation of GATC sites in pBR322 DNA by Dam methyltransferase from Escherichia coli

The methylation pattern of the 22 GATC sites of pBR322 (dam-) by Dam methyltransferase from Escherichia coli has been studied. Preferential hemimethylation took place at positions 3042 and 349. It was found that these preferential methylations were the same in supercoiled circular and linear DNAs. The flanking regions of these preferentially methylated sites contain three G.C pairs on one side and two A.T pairs and one G.C pair on the other. This preferential methylation was confirmed on a 126-base pair oligonucleotide containing two GATC sites with different flanking sequences. The next sites methylated were, in both cases, the first GATC site on the A.T-rich side, although the orientation was different. The rapid methylation of a second and third neighboring GATC site on the same plasmid suggests a processive mechanism. The implications of the orientation of hemimethylation are discussed in the context of the recognition of a palindromic target site by a monomeric DNA-binding protein.     

Cooperativity in nucleosomes assembly on supercoiled pBR322 DNA.

Many studies have shown that in reconstituted chromatin model systems, containing only purified DNA and histone octamer, nucleosomes can adopt well defined locations with respect to DNA nucleotide sequence. Recently, nucleosome-nucleosome interactions were suggested as one of the factors underlying preferential nucleosomes positioning. In the present paper this aspect has been studied by topological analysis and electron microscopy visualization of minichromosomes reconstituted at different histone/DNA ratios. Both methods suggest that cooperativity plays a role in nucleosomes formation. A linear cooperative model in which nucleosomes are formed on discrete sites with cooperative interactions occurring only between nearest neighbours allows to calculate the cooperative constant. The reported results show that basic interactions, which are of relevance in the process of chromatin folding, are present also in very simple model system.     

Supercondensed structure of plasmid pBR322 DNA in an Escherichia coli DNA topoisomerase II mutant

An unusual structural component, supercondensed pBR322 DNA, has been found in plasmid pBR322 DNA samples isolated from a DNA topoisomerase II mutant of Escherichia coli, SD108 (topA+, gyrB225). The supercondensed pBR322 DNA moved faster than supercoiled pBR322 DNA as a homogeneous band in agrose gels when the DNA samples were analysed by electrophoresis. The mobility of the supercondensed DNA was not substantially affected by chloroquine intercalation. The supercondensed pBR322 DNA migrated as a high density "third DNA band" when the samples were subjected to caesium chloride/ethidium bromide gradient equilibrium centrifugation. The unusual pBR322 DNA visualized by electron microscopy was a globoid-shaped particle. These observations suggest that the pBR322 plasmid can assume a tertiary structure other than a supercoiled or relaxed structure. DNA topoisomerases may be involved in the supercondensation of plasmid DNA and chromosomal DNA.     

Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

Abstract We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli relA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yield per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli .     

DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli

The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but -2 frameshifts and +1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G.C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.     

Adaptation of Escherichia coli growth rates to the presence of pBR322

Changes in the growth rate of Escherichia coli K12 J62-1 in response to the presence of plasmid pBR322 have been investigated. Plasmid-free and plasmid-containing strains were grown in batch culture and their maximum specific growth rate (μ_max) determined. The acquisition of pBR322 by the host resulted in a decreased μ_max. Following repeated subculturing of the plasmid-containing strain on selective medium, restoration in μ_max was observed. The copy number and structure of the plasmid were not significantly altered during the experiment Growth rate measurements for a series of strains constructed using a combination of host cells and plas-mids with and without culture histories, indicated that the site of the adaptive mutation was located on the host chromosome rather than on the plasmid.     

Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli.

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.     

Maintenance stability of the pBR322 and pBR327 vector plasmids in Escherichia coli cells during multiple passages

Stability of pBR322 and pBR327 plasmids was studied. Plasmid-containing Escherichia coli strains were grown in liquid growth medium without selection pressure. Plasmid pBR327 was shown to be more stable in E. coli CSH54 cells than pBR322. Essential heterogenity of individual plasmid-containing clones was recognized by the maintenance stability of plasmid DNA. The indicated clones with high stability failed to be cured from pBR327 plasmid by means of acridine orange. High stability of plasmid maintenance and the failure to cure cells containing this plasmid are suggested to correlate with and to be essentially determined by the cell functions.     

Persistence of pBR322-related plasmids in Escherichia coli grown in chemostat cultures

Abstract The stability of plasmid pBR322 and a number of close derivatives was examined by continuous culture of Escherichia coli . Cultures were subjected to either glucose, phosphate or magnesium limitation in non-selective medium at a dilution rate of 0.1/h. Under these conditions pBR322 was eventually lost from the population, but only after a distinct lag period. The closely related plasmids pBR325 and especially pBR327 and pBR328, but not pAT153, were lost more rapidly. Three cosmids pHC79, pSJ55 and pJB8 were generally found to be less stable than the pBR322-type plasmids from which they were derived. Chimaeric plasmids containing DNA from yeast and from a thermophilic bacillus were also unstable in E. coli .     

Synthesis of RNA I by the RNA polymerase from Micrococcus luteus on the Escherichia coli plasmid pBR322

A mutant strain of Bacillus megaterium, arising spontaneously and resistant to micrococcin , possesses ribosomes which contain an altered form of protein BM-L11 (the homologue of Escherichia coli protein L11). Reconstitution analysis has revealed that the alteration to protein BM-L11 is the sole cause of resistance in this strain.     

Stability of ColE1-like and pBR322-like plasmids in Escherichia coli

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1-like and pBR322-like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid-free and of plasmid-carrying cells, assuming that the units of partition are distributed randomly between daughter cells. Stability of the pBR322-like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322-like plasmids, present mainly as dimers, and the ColE1-like plasmid showed greater stability than that predicted with random partitioning at cell division.