LEADING AND LAGGING STRAND DNA SYNTHESIS IN EXTRACHROMOSOMAL ELEMENTS OF E. COLI

Abstract

Plasmids containing the origin of (PHI)X174 leading strand DNA synthesis and pBR322 DNA were shown to function as templates in (PHI)X174 DNA replication reactions in vitro. These plasmids were: (i) cleaved by the (PHI)X174 A protein; (ii) supported net synthesis of unit length single stranded circular DNA in the presence of the (PHI)X174 A protein and the Escherichia coli rep protein, DNA binding protein, and the DNA polymerase III elongation system; and (iii) supported duplex DNA replication catalyzed by the (PHI)X174 A protein and crude extracts of E. coli. These data suggested that lagging strand origins of DNA replication were present on each strand of pBR322 DNA. A DNA sequence on each strand of pBR322 was shown to function as an effector site for E. coli replication factor Y ATPase activity. This site specific ATPase hydrolysis had been previously correlated with (PHI)X174 lagging strand DNA synthesis. No sequence homology was observed between the two pBR322 and the single (PHI)X174 factor Y effector sites. In contrast to bacteriophage fl DNA, recombinant phage fl DNA containing the factor Y sites of pBR322 were conveted in vitro to the duplex DNA form in a (PHI)X-like mechanism which required the E. coli dna B, dna C, and the dna G gene products and was resistant to rifampicin.