EVALUATION OF ESCHERICHIA-COLI RECBC SBCBC MUTANTS FOR CLONING BY RECOMBINATION IN-VIVO

In vivo recombination as a tool for plasmid construction was analyzed using a model system based on the properties of the RecF pathway in Escherichia coli. This pathway was used after a double strand break (DSB) effected by restriction enzyme cleavage of the plasmids of interest. DSB repair was shown to be independent of the methylation state of the vector or insert DNA, of the type of restriction enzyme used (5' or 3' overhanging or blunt ends) and of dephosphorylation of the vector and/or template. Since the E. coli repair system does not recognize insertions, this recombination mechanism can be used to exchange cDNAs between different vectors. Some implications of the results on the mechanism of recombination are discussed.