The recF-dependent endonuclease from Escherichia coli K12. Formation and resolution of pBR322 DNA multimers

Abstract

The instability of supercoiled pBR322 DNA obtained from different cells has been investigated. Partially purified plasmid DNA species from rec+, recA and recBC sbcB cells are converted in vitro first to relaxed and then to linear molecules. The recA and recBC sbcB cells produce the best conditions for the monomerization of the pBR322 DNA and the stable maintenance of plasmids. The supercoiled pBR322 DNA from the recBC sbcB recF144 cells has been isolated preferentially in multimeric form (circular oligomers). These DNA forms are not converted to plasmid monomers and are converted to linear molecules three-fold slower than the monomer linearization in the case of the recBC sbcB cells. On the other hand, incubation of the pure pBR322 DNA with the recF-dependent protein Z (Krivonogov and Novitskaja 1982) results in the ATP-independent conversion of supercoiled plasmid DNA to relaxed and linear molecules. These results demonstrate an endonuclease activity of the recF-controlled protein Z, which may be involved in general recA-dependent recombination and formation of the pBR322 monomers in the cell. The results also show that the recF144 mutation in recBC sbcB recF and recF cells leads to the absence of detectable amounts of a 49,000 molecular weight protein.