Journal of Molecular Biology
Replication of bacteriophage DNA: I. Replication of DNA of lambda phage defective in early functions☆
Abstract
DNA synthesis of early sus (suppressor sensitive) mutants of phage λ in su− cells was studied by the density-gradient centrifugation method. The functions of the O and P cistrons are essential for replication of the phage genome while the function of the N cistron is not. DNA molecules of susN mutants replicate at a slow rate. A majority of the molecules replicate more than once. For normal replication to occur the N function has to be expressed.
The functions of the O and P cistrons are expressed in the absence of the N function. Superinfection of a heteroimmune lysogen by a susP mutant fails to elicit the function required for replication of the superinfecting phage genome.
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Forty temperature-sensitive mutations affecting lytic growth and eight affecting both establishment and maintenance of lysogeny of the temperate mycobacteriophage L1 have been isolated. All of the latter mutations form one complementation group and map within a very short region around the 15% coordinate of the L1 genome; these affect a single gene, cl, coding for the L1 repressor. The former 40 mutations form 28 complementation groups, identifying 28 different genes, G1-G28, essential for the lytic growth of L1. These genes have been mapped using the Gts mutations. Of the 28 Gts mutants, 14 are defective in host lysis at 42° but not at 32° while the other 14 can lyse the host at both temperatures. Among the former 14 Gts mutants, 6 are also defective in L1 DNA synthesis at 42°, and they map in two different clusters, 4 around 65% and 2 around 84% of the L1 genome.
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Regulation of βdv plasmid DNA replication. A quantitative model for control of plasmid βdv replication in the bacteriol cell division cycle
1986, Journal of Molecular BiologyA quantitative model for the regulation of replication of plasmid βdv in the Escherichia coli cell division cycle has been developed. The regulatory processes include the interactions of cro repressor proteins with the rightward operator DNA sites, the transcriptional activation of the βdv replication origin, and the interaction of initiation proteins with activated origins to form functional replication initiation complexes. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to the events of the cell cycle such as initiation of mRNA transcription and protein translation and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and repressor and initiator protein concentrations of the model agreed with experimentally determined estimates. Simulated deviations from regular segregation of the various components at cell division, such as plasmid copies and free and bound repressor proteins, suggest that βdv replication control responds only slowly to these perturbations. The consequence of this slow response to perturbations, which are expected at a random frequency, was simulated for a population of βdv-containing cells in a growing culture. This simulation predicts instability of inheritance of βdv plasmids in the population, despite the very high plasmid copy number, in agreement with experimental observation.
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The ‘groplaque’ (plaque-shaped growth spot) assay, which selects for CmR growth in an MS 1449 lawn at 32°C after an initial plaquing period at 37°C, reveals two distinguishable classes of pλCM isolates. All variants whose deletions extend into or beyond rexB give rise to visible CmR growth only after the temperature shift to 32°C, and thus produce a hollow-centered ‘donut’ type of groplaque. In contrast, 16 out of 17 variants whose deletions fall short of rexB produce ‘solid’ groplaques which appear before the temperature shift. Tests of T4rII phage exclusion show the exceptional 17th variant to be Rex−, confirming the identification of rex as the λ component whose loss results in the ‘donut’ groplaque morphology.
More specific physiological tests showed that in the absence of Rex the establishment of a newly injected pλCM plasmid becomes temperature-sensitive (ts), while plasmid maintenance remains unaffected. This indicates that the role of Rex in plasmid survival is confined to the early stages of transduction, where it might either assist plasmid replication or retard host replication, to help the plasmid replicon achieve a copy number sufficient for stable transmission.
The role of the product of gene O of bacteriophage λ in phage DNA replication was examined by shifting cells infected with an Ots mutant to the nonpermissive temperature after incubation at the permissive temperature. Thymidine incorporation after the temperature shift exhibits biphasic kinetics, with rapid synthesis immediately after the shoft and slower synthesis 2–15 min after the shift. Following a shift to the nonpermissive temperature early in infection, the proportion of replicative intermediates decreases substantially and σ-structures are favored for preservation. When the shift is done late in infection, the proportion of replicative intermediates remains the same. The average length of single-stranded regions at the branch points increases after a shift to the nonpermissive temperature. Most of the counts which are incorporated after the temperature shift are incorporated into strands which are longer than unit length. These results favor a model in which λ O protein is required for the initiation of replication, but at least some elongation can continue in the absence of O. It is possible that O protein plays a role in elongation of the lagging strand at replicative forks. This model suggests a way to regulate the transition between θ and σ replication which occurs as λ infection proceeds.
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This work was aided in part research grant GM-08384 from the United States Public Health Service.