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

Abstract

There are two 'pathways' of replication of lambda 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 lambda 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.

Figure 1

Replication of the λ plasmid, pKB2, in amino acid-starved relA2 (A) and relA⁺ (B) hosts. Bacteria were grown in a minimal medium, isoleucine starvation was induced at time 0 and samples of equal cell mass (1 OD unit) for total DNA isolation were withdrawn at 1, 2 and 3 h after onset of the starvation. Numbers above lanes represent these times. Plasmid monomers and dimers, and chromosomal DNA bands are indicated as P-m, P-d and Ch, respectively.

Figure 2

Map of the λ plasmid, pKB2, used in this work with indicated restriction sites and fragments used as probes (external circles) in 2D-AGE and employed in electron microscopic studies.

Figure 3

Theoretical schemes of expected pictures of 2D-AGE of λ plasmid (pKB2), digested with HindIII and BamHI, replicating according to unidirectional leftward θ mode (Uni-L), unidirectional rightward θ mode (Uni-R), bidirectional θ mode (Bi) and a combination of molecules replicating according to all these modes (All). The schemes were obtained using a computer model for the analysis of DNA replication intermediates by 2D-AGE (31), and considering particular pictures, which might appear after digestion of pKB2 replication intermediates with certain restriction enzymes followed by 2D-AGE.

Figure 4

2D-AGE analysis of directionality of replication of the λ plasmid, pKB2, in non-starved relA⁺ cells (A), non-starved relA2 mutant (B) and isoleucine-starved relA2 mutant (C and D). The experiments in which HindIII and BamHI (A–C) or NdeI (D) digestions were performed are shown. Left panels represent autoradiograms and right panels show their interpretation. ‘Uni-R’ and ‘Uni-L’ denote dots characteristic for rightward and leftward unidirectional replication, respectively. Positions of DNA molecules of particular shapes are indicated by arrows.

Figure 5

Overexposure of the gel obtained after 2D-AGE of the λ plasmid, pKB2, isolated from isoleucine-starved relA2 cells and digested with HindIII and BamHI (compare with Fig. 4C). The left panel represents the autoradiogram and the right panel shows its interpretation (analogously to pictures presented in Fig. 4). The juxtaposed but not coincidental bubble arcs are visible; however, other signals are too strong to be analyzed. Very similar patterns were obtained when plasmid pKB2 was isolated from non-starved relA⁺ and relA2 cells (data not shown).

Figure 6

Examples of replication intermediates of the λ plasmid, pKB2, analyzed under an electron microscope (A–C) and a gallery of representative results obtained after measurement of lengths of fragments of DNA molecules (D). Following isolation of plasmid DNA from isoleucine-starved relA2 cells and digestion with BamHI, samples were prepared for electron microscopy. Examples of replication intermediates, which contain bubbles indicating unidirectional (A) and bidirectional (B and C) DNA replication, are presented. Positions of oriλ regions are shown by arrows. Bar represents 1 kb. Schemes of representative replication intermediates (grouped according to directionality of replication) are presented below the map of the investigated region with the oriλ region marked (D). Bi, bidirectional replication; Uni-R, unidirectional rightward replication; Uni-L, unidirectional leftward replication.