Maximal limits of the Escherichia coli replication factor Y effector site sequences in pBR322 DNA

Abstract

pBR322 DNA contains two separate regions on opposite strands and close to the origin of replication which, when in single-stranded form, can act as effectors for the ATPase activity of Escherichia coli replication factor Y. Small fragments of DNA containing these sites when cloned into an fl phage vector act as origins of DNA replication allowing the formation of complementary double-stranded DNA in a rifampicin-resistant, dnaB-, dnaG-, and dnaC-dependent fashion in vitro. We report here the maximal limits of the E. coli replication factor Y effector sites of pBR322 DNA. The site on the H strand of pBR322 DNA can form a structure resistant to digestion by E. coli exonuclease VII, the site on the L strand cannot. The H and L strand sites lie within nucleotides 2144-2185 and 2353-2416, respectively, of pBR322 DNA. A deletion of 34 nucleotides (nucleotides 2121-2154) within the H strand site renders it totally inactive as an effector for factor Y ATPase activity. No extensive homology could be detected between the three known factor Y effector sites, the two reported here and the one previously identified on the phi X174 viral DNA strand (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 799-803).