Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity.
rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.[1]References
- Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity. Subia, N.L., Kogoma, T. J. Mol. Biol. (1986) [Pubmed]
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