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Lethal effect of λ DNA terminase in recombination deficient Escherichia coli

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Summary

λ DNA terminase is the enzyme that catalyses the cleavage of λ DNA concatemers into genome-size molecules and packages them into the capsid. The cleavage (DNA maturation) takes place in a specific site in the phage DNA called cos. Either one of two Escherichia coli proteins, integration host factor (IHF) and terminase host factor (THF), is required, in addition to terminase, for maturation of wild-type λ DNA in vitro. In vivo, at least some cos cleavage is known to occur in mutants that are unable to synthesize active IHF. No THF-defective mutants have yet been isolated. In order to determine if IHF, THF or any other host protein is involved in λ DNA maturation in vivo, I devised a selection for host mutants that are unable to support cos cleavage. The selection is based on the assumption that λ DNA terminase will kill cells by cleaving chromosomally located cos sites. I found that DNA terminase will indeed kill cells provided that they contain a chromosomal cos site and provided also that they are defective in the host recA or recB genes. These two genes are required for certain pathways of genetic recombination and repair of damaged DNA, and I suggest that they prevent terminase-induced killing by repairing broken chromosomes. Interstingly, mutation in a related host gene, recD, did not render cells susceptible to terminase killing. recD and recB both encode subunits of exonuclease V, but recD mutants, unlike recB, remain proficient in genetic recombination and repair. I found mutants that survived the lethal effect of terminase in cos-containing E. coli recA at a frequency of about 5×10-5. About 90% of these survivors were defective in terminase synthesis, and the rest were defective in IHF function. This result suggests that in the absence of IHF in vivo cos cleavage decreases to a level that permits repair of the damage, and therefore survival, even in recombination deficient cells. The absence of mutations in any other host gene suggests that IHF is the major accessory factor in λ DNA maturation in vivo. Alternatively, or in addition, mutations in other accessory factors are lethal.

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Abbreviations

gp:

gene product: e.g. gpA, product of gene A

():

prophage state

[]:

plasmid-carrier state

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Authors and Affiliations

  1. Department of Medical Genetics, University of Toronto, M5S 1A8, Ontario, Canada

    Helios Murialdo

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  1. Helios Murialdo
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Communicated by G.R. Smith

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Murialdo, H. Lethal effect of λ DNA terminase in recombination deficient Escherichia coli . Molec. Gen. Genet. 213, 42–49 (1988). https://doi.org/10.1007/BF00333396

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