DNAK HEAT-SHOCK PROTEIN OF ESCHERICHIA-COLI MAINTAINS THE NEGATIVE SUPERCOILING OF DNA AGAINST THERMAL-STRESS

Plasmid DNA in exponentially growing Escherichia coli immediately rela xes 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 hea t shock protein off. coli, a prokaryotic homologue of hsp70, is involv ed in this re-supercoiling of DNA. As re-supercoiling of DNA did not o ccur 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-supercoili ng of DNA was not observed. DNAs incubated with a crude cell extract p repared from the dnaK mutant were more relaxed than seen with the extr act from its isogenic wild-type strain, and the addition of purified D naK protein to the mutant extract led to an increase in the negative s upercoiling 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 maintaini ng the negative supercoiling of DNA against thermal stress.