Stimulation of adenosine 5'-triphosphate-dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli

Abstract

In vitro deoxyribonucleic acid (DNA) synthesis systems based on an earlier system using penicillin have been developed which use osmotic lysis of lysozyme-formed spheroplasts of Escherichia coli cells embedded in an agarose matrix. An adenosine 5'-triphosphate (ATP)-dependent semiconservative mode, or replicative mode, of in vitro DNA synthesis is exhibited which is sensitive to nalidixic acid. These systems require growth of the agar-embedded cells in a preincubation medium before spheroplast formation and osmotic lysis. Inhibitor studies suggest that one or more required macromolecular species are synthesized during this preincubation growth period. Osmotic shock fluid from E. coli contains macromolecular factors which preferentially stimulate the ATP-dependent semiconservative mode of in vitro DNA synthesis. In some cases, the ATPindependent mode of synthesis is inhibited by shock fluid. Evidence is presented that the stimulating factors found in the osmotic shock fluid come from the E. coli periplasmic space. This stimulation is observed using either toluene-treated cells or lysed agar-embedded ethylene glycol-bis-(beta-aminoethyl ether) N,N'tetraacetate-lysozyme spheroplasts, and is thus independent of the in vitro DNA synthesis system used. Shock fluid obtained from a given E. coli dna mutant does not stimulate in vitro DNA synthesis by that mutant. However, in some cases, shock fluid from one class of dna mutants does stimulate ATP-dependent in vitro DNA synthesis by another class of dna mutants, in a thermosensitive reaction. Gently prepared cell extracts also stimulate ATP-dependent in vitro DNA synthesis, whereas cell extracts prepared by more severe procedures inhibit this in vitro synthesis. Several stimulating DNA replication factors may be present in the osmotic shock fluid, including products of E. coli dna genes.Characteristics of an in vitro deoxyribonucleic acid (DNA) replication system using Escherichia coli cells immobilized in a matrix of agarose have been described (33,34). DNA synthesis in this in vitro system closely resembles DNA replication in vivo in an adenosine 5'-triphosphate (ATP)-dependent process, as shown by the criteria of semiconservative synthesis, reduced synthesis in E. coli dna mutants (mutants thermosensitive for DNA replication [10]), and inhibition of in vitro DNA synthesis by specific inhibitors of DNA replication. In a similar DNA synthesis system using cells immobilized on a planar surface of cellophane, Schaller et al. (28) have shown that one or more nondialyzable components must be present at in vivo concentrations for replicative in vitro DNA synthesis to proceed. The agarose matrix 'Present address: Department of Biochemistry, University of Massachusetts, Amherst, Mass. 01002. thus appears to serve two purposes: (i) it maintains the intactness of the fragile DNA replication complex of DNA, intracellular site, and other components (31, 33); and (ii) it retains certain macromolecular components essential for replicative in vitro DNA synthesis at the required high c...