Selective cloning of ColE1 DNA initiation sequences using the cloning vector M13ΔE101

Abstract

DNA sequences required to direct single-strand to double-strand conversion by a øX174-type mechanism have been detected in ColEl plasmid DNA. The sites responsible for this DNA strand initiation, named rriA and rriB, have been localized to the L strand of the HaeII-E fragment and to the H strand of the HaeII-C fragment of ColE1. To define rriA and rriB more precisely, random fragments of ColEl DNA have been cloned into the single-stranded phage cloning vector M13ΔE101. This phage contains a viable deletion of the M13 complementary strand origin and makes small turbid plaques. Cloning of DNA initiation determinants into the unique EcoRI site of M13ΔE101 rescues the mutant phage and restores the ability to form clear plaques. DNA sequence analysis of the random, 100–400 bp inserts in clear plaque isolates has localized the rriA and rriB determinants within DNA fragments of about 100 bp. These initiation determinants promote assembly of a functional, multiprotein priming complex, “primosome”, identical to that utilized in the in vitro conversion of φX174 single-stranded DNA to the duplex replicative form.