DnaK Heat Shock Protein of Escherichia coli Maintains the Negative Supercoiling of DNA against Thermal Stress

Abstract

Plasmid DNA in exponentially growing Escherichia coli immediately relaxes after heat shock, and the relaxed state of DNA rapidly reverts to the original state with exposure to conditions of heat shock. We have now obtained genetic and biochemical evidence indicating that DnaK heat shock protein of E. coli, a prokaryotic homologue of hsp70, is involved in this re-supercoiling of DNA. As re-supercoiling of DNA did not occur in an rpoH amber mutant, it seems likely that heat shock proteins are required for this reaction. Plasmid DNA in a dnaK deletion mutant relaxed excessively after temperature shift-up, and the re-supercoiling of DNA was not observed. DNAs incubated with a crude cell extract prepared from the dnaK mutant were more relaxed than seen with the extract from its isogenic wild-type strain, and the addition of purified DnaK protein to the mutant extract led to an increase in the negative supercoiling of DNA. Moreover, reaction products of purified DNA gyrase more negatively supercoiled in the presence of DnaK protein. Based on these results, we propose that DnaK protein plays a role in maintaining the negative supercoiling of DNA against thermal stress.