Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli

Summary

Monomeric pBR322 DNA that had been linearized at its unique SalI site transformed wild-type Escherichia coli with 10² to 10³ times less efficiency than CCC plasmid DNA. Dose-response experiments indicated that a single linear plasmid ‘molecule’ was sufficient to produce a transformant. Transformation with linearized pBR322 DNA was reduced 10 to 40 fold in recA ⁻, recBC ⁻ or recF ⁻ backgrounds. In contrast, transformation with CCC DNA was unaffected by the rec status of the host. Transformation with linear pBR322 DNA was increased 3-fold in a DNA ligase-overproducing (lop11) mutant and decreased to a similar degree by transient inactivation of ligase in a ligts7 mutant.

A proportion (ranging from about 9% in the wild-type to 42% in a recBC, lop11 mutant) of the transformants obtained with SalI-linearized pBR322 monomeric DNA contained deleted plasmids. Deletion rates were generally higher in rec ⁻ strains. Dephosphorylation of the termini on linear DNA or the creation of blunt-ended pBR322 molecules (by end-filling the SalI 5′ protrusions or by cleavage with PvuII) decreased the transformation frequencywhilst increasing the deletion rate.

Linear pBR322 dimeric DNA gave transformation frequencies in recA ⁺ and recA ⁻ strains that were reduced only 3 to 7 fold respectively relative to frequencies obtained with dimeric CCC DNA. Furthermore, in contrast to transformation with linear monomeric DNA, deletions were not observed.

We propose that the majority of transformants arise, not by simple intracellular reannealing and ligation of the two cohesive SelI-termini of a linear molecule, but by intramolecular recombination. Deleted plasmids could be generated therefore during recyclization caused by recombination between short directly repeated sequences within a pBR322 monomer. We suggest that perfectly recircularized monomeric pBR322 molecules, which are found in the majority of transformants, arise primarily by intramolecular recombinational resolution of head-to-tail linear pBR322 dimers. Such linear oligomeric forms are created during preparation of linearized plasmid DNA by annealing of the SalI cohesive termini and constitute a variable proportion of the total molecules present.