Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication.

Abstract

We examined the role of two Escherichia coli heat shock proteins, the dnaK and dnaJ gene products, during the initiation of .lambda.dv DNA replication in vitro. Using 14C-labeled .lambda. P protein we showed that the DnaK and DnaJ heat shock proteins function together to release .lambda. P protein from the preprimosomal complex consisting of .lambda. origin of replication-.lambda. O-.lambda. P-DnaB protein. Hydrolysis of ATP, catalyzed presumably by DnaK, is required during this reaction. Substitution of DnaK protein with that of the mutant DnaK756 protein blocks .lambda. P release. After DnaK and DnaJ action, the preprimosomal complex, isolated on Sepharose 4B, can support .lambda. dv DNA replication without any additional prepriming proteins. Using DnaK-affinity chromatography we showed that both .lambda. O and .lambda. P proteins bind to DnaK protein. The .lambda. P protein interacts with DnaK protein in a salt-resistant, hydrophobic manner, and ATP hydrolysis is necessary to elute at least part of .lambda. P protein from the DnaK-affinity column. The proposed mechanism of action of the prokaryotic DnaK and DnaJ heat shock proteins agrees with the hypothesis that Hsp70, the DnaK analogue of eukaryotes, uses ATP to disrupt hydrophobic aggregates [Pelham, H. R. B. (1986) Cell 46, 959-961].