[19] DNA Synthesis in Toluene-Treated Cells of Escherichia coli

Toluene treatment kills ceils of Escherichia coli and makes them permeable to molecules of low molecular weight. Despite the fact that these cells are no longer viable, they retain a number of their physiological functions and the ability to synthesize DNA, if supplied with the necessary substrates. This chapter describes the assay of DNA synthesis in toluene-treated cells based upon the incorporation of radioactively labeled deoxynucleotide residues into acid-insoluble polydeoxynucleotides. DNA synthesis in toluene-treated cells occurs in a normal manner if the reaction mixture contains Triton X-100, a nonionic detergent. One of the convincing arguments that DNA synthesis in the toluene-treated cell system duplicates active replication in vivo has been the temperature effect on temperature-sensitive replication mutants in vitro.