Two distinct mechanisms of synthesis of DNA fragments on colicin E1 plasmid DNA.

Abstract

REPLICATION of closed-circular colicin E1 plasmid (Col E1) DNA can be initiated and completed in extracts of Escherichia coli1,2. The major products of in vitro replication are completely replicated molecules (23S) and a unique type of early replicative intermediate (26S) containing a newly synthesised DNA fragment(s) in a small replication loop. The fragment has an average length of approximately one fifteenth of the unit length of the plasmid molecule and has a sedimentation constant of approximately 6S1,3. The replicated region of the intermediate consists of either one double-stranded branch and one single-stranded branch (type I) or two double-stranded branches (type II)2,4. These intermediates accumulate in a reaction mixture containing 10% glycerol2,3. Synthesis of the intermediates is inhibited by rifampicin1,3 but most of the intermediates can complete replication in the presence of rifampicin3. Rifampicin is also known to inhibit in vivo replication of Col E1 DNA5. We have studied the synthesis and fate of 6S DNA fragments formed on the parental heavy (H) strands and those formed on the parental light (L) strands (defined by CsCI density gradient centrifugation in the presence of poly (U,G)) of early replicative intermediates. The results show that the first synthesis of a DNA fragment is initiated at a specific site on the H strand and depends on the function of E. coli DNA-dependent RNA polymerase. Subsequent synthesis of the DNA fragment on the L strand does not involve the RNA polymerase.