Persistence of the pBR 322 plasmid in Escherichia coli K 12 grown in chemostat cultures

Populations of a Escherichia coli K 12 strain, containing the vector plasmid p BR 322, were grown in chemostat culture under glucose- and phosphatelimited conditions. Resistance to tetracycline and ampicillin were lost after prolonged cultivation, resulting in the production of apparent plasmid-free populations which were more competitive than the original population. This competitiveness between plasmid-free and plasmid-containing populations was greatest in environments where the nutrient restriction was severe. Also during sequential subcultivation in batch cultures loss of plasmid was observed.     

Processing of G4 DNA by Dna2 helicase/nuclease and replication protein A (RPA) provides insights into the mechanism of Dna2/RPA substrate recognition

The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5'- to 3'-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3'- to 5'-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening.     

GATC motifs may alter the conformation of DNA depending on sequence context and N6-adenine methylation status: possible implications for DNA-protein recognition

As part of our analysis of the role of a uniquely clustered set of dam methylation sites (the motif GATC) within the origin of DNA replication in Escherichia coli, we have studied the effect of GATCs in various methylation states on the intrinsic curvature of DNA. We have designed a set of DNA linkers and used commercially available linkers containing GATC motifs. The linkers were ligated and the electrophoretic mobility of the resulting multimers in different states of methylation was tested relative to reference fragments. We report that properly phased GATCs in certain sequence environments modulate DNA curvature and that these effects may be enhanced by N⁶-adenine methylation of the GATCs. These structural alterations may in turn affect DNA-protein interactions, especially those involving proteins that rely on both primary sequence and structure for recognition. We present an example, where introduction of a GATC within an integration host factor (IHF) binding site, which does not alter the consensus sequence, reduces the binding affinity of the protein for the modified site. Received: 16 December 1997 / Accepted: 24 February 1998     

Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented.     

Acetylation of Dna2 Endonuclease/Helicase and Flap Endonuclease 1 by p300 Promotes DNA Stability by Creating Long Flap Intermediates

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication ( oriC  ) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC . GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis -acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.