SPECIFIC CLEAVAGE OF CHROMOSOMAL AND PLASMID DNA STRANDS IN GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA CAN BE DETECTED WITH NUCLEOTIDE RESOLUTION

A sensitive and precise in vitro technique for detecting DNA strand di scontinuities produced in vivo has been developed. The procedure, a fo rm of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of str and scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coil. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positi ons of the chromosomally integrated fertility factors were also observ ed. Detection of DNA strand scission at the nic position of F DNA in t he high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable el ement, but was independent of mating. These results imply that not onl y the transfer origins of extrachromosomal F and F-like fertility fact ors, but also the origins of stably integrated copies of these plasmid s, are subject to an equilibrium of cleavage and ligation in vivo in t he absence of DNA transfer.