Directionality of lambda plasmid DNA replication carried out by the heritable replication complex

There are two ‘pathways’ of replication of λ plasmids in Escherichia coli. One pathway requires the assembly of a new replication complex before replication and the second pathway is based on the activity of the replication complex inherited by one of two daughter plasmid copies after a preceding replication round. Such a phenomenon was postulated to occur also in other replicons, including Saccharomyces cerevisiae autonomously replicating sequences. Here we investigated directionality of λ plasmid replication carried out by the heritable and newly assembled replication complexes. Using two-dimensional agarose gel electrophoresis and electron microscopy we demonstrated that in both normal growth conditions and during the relaxed response to amino acid starvation (when only replication carried out by the heritable complex is possible), bidirectionally and undirectionally replicating plasmid molecules occurred in host cells in roughly equal proportions. The results are compatible with the hypothesis that both complexes (heritable and newly assembled) are equivalent.     

Random replication of the stringent plasmid R1 in Escherichia coli K-12.

The R-factor R1 is present in a low number of copies per genome (near unity, so-called stringent control of replication). The replication of R1 was studied in a density-shift experiment. One generation after the shift about 20% of the R1 copies had not replicated, whereas about 20% had replicated at least twice. The results are in quantitative accordance with a random replication of R1 in which the replicating molecules are taken from a cytoplasmic plasmid pool and transferred back to the pool after replication. This is analogous to the results obtained by Bazaral and Helinski (1970) and Rownd (1969) for plasmids that are present in 10 to 20 copies per genome (so-called relaxed control of replication). Hence, there seem to be no difference between stringent and relaxed plasmids with respect to selection of plasmid molecules for replication. However, we cannot tell whether all R1 copies in a cell replicate during a fraction of or throughout the cell cycle. The random selction of plasmid copies for replication has to be considered when models for control of replication are constructed.     

Stability of coliphage lambda DNA replication initiator, the lambda O protein.

The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.     

The control of lambda DNA terminase synthesis.

Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques.     

Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins

Using highly purified bacteriophage lambda and E. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid DNA. The addition of a new E. coli factor, the grpE gene product, to this replication system reduced the level of dnaK protein required for efficient DNA synthesis by at least 10-fold, and also allowed the isolation of a stable DNA replication intermediate. Based on all available information, we propose a molecular mechanism for the action of the dnaK and grpE proteins during the prepriming reaction leading to lambda DNA synthesis.     

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.     

P1 plasmid replication requires methylated DNA.

Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.     

Intermediates in plasmid pT181 DNA replication.

Staphylococcus aureus plasmid pT181 is thought to replicate via an asymmetric rolling-circle mechanism. By studying pulse labeled replicative intermediates, here we report that pT181 replication involves: (1) a post-replicative hypersupercoiled monomer and (2) a partially replicated intermediate which lacks superhelicity but is unlike a typical rolling-circle intermediate in that only nascent strands of less than unit length are released by alkali denaturation. A model for pT181 replication is proposed to accommodate this apparent discrepancy.     

Inheritance of the replication complex by one of two daughter copies during lambda plasmid replication in Escherichia coli.

Direct measurement of DNA synthesis confirmed that lambda plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is lambda cro-independent, but requires the function of lambda O initiator in the absence of its synthesis. This suggests that after the assembly of the replication complex (RC) at ori lambda the lambda O protein remains in this structure and the affinity of lambda O to ori lambda is alleviated in the assembled RC allowing its movement along the DNA. During amino acid starvation the lambda plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant. This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round. Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication. We infer that this is the plasmid copy that inherits the lambda O-enclosing RC from the previous replication round. Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation. Therefore, we presume that in lambda plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication. This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC. In the absence of lambda O initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC. Our results are discussed in relation to the model of regulation of lambda plasmid replication.     

The bacteriophage lambda O and P protein initiators promote the replication of single-stranded DNA.

A soluble enzyme system that specifically initiates lambda dv plasmid DNA replication at a bacteriophage lambda replication origin [Wold et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6176-6180] is also capable of replicating the single-stranded circular chromosomes of phages M13 and phi X174 to a duplex form. This chain initiation on single-stranded templates is novel in that it is absolutely dependent on the lambda O and P protein chromosomal initiators and on several Escherichia coli proteins that are known to function in the replication of the lambda chromosome in vivo, including the host dnaB, dnaG (primase), dnaJ and dnaK replication proteins. Strand initiation occurs at multiple sites following an O and P protein-dependent pre-priming step in which the DNA is converted into an activated nucleoprotein complex containing the bacterial dnaB protein. We propose a scheme for the initiation of DNA synthesis on single-stranded templates in this enzyme system that may be relevant to strand initiation events that occur during replication of phage lambda in vivo.     

An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control

Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.