The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system.

Abstract

The Escherichia coli dnaK gene product, originally defined by mutations that blocked A phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The A 0 and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified A P protein and stimulated by purified A 0 protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the 0 and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.The study of host-virus interactions has been greatly facilitated through the isolation of host mutants that block viral growth. Through the use of indirect or direct selections, various investigators have uncovered five Escherichia coli genes whose products are necessary for bacteriophage A DNA replication: dnaB, dnaK, dnaJ, grpD, and grpE (1-4). Of these genes, only dnaB has been convincingly shown to be essential for E. coli DNA replication (5). Both the dnaK+ and dnaJ+ gene products are essential for bacterial growth as well, because some mutations in these genes block colony formation at 42°C. Both DNA and RNA bacterial metabolism are affected in dnaK-(4, 6) and dnaJ-mutants (7,8). The effect on RNA metabolism is unique and not shared by mutations in the rest of the dna genes of E. coli (5). Mutations in A phage that enable it to grow on dnaKhosts map in the P gene, which suggests an interaction between the host dnaK protein and the phage P protein (1, 2). The dnaK gene has been cloned into A phage (2), and its gene product has been identified by NaDodSO4/polyacrylamide gel electrophoresis (9) and shown to be identical to protein B66.0 (10), one of the heat shock proteins of E. coli (11)(12)(13). In this communication, we report that the dnaK+ protein (i) possesses a weak DNA-independent ATPase activity that is modulated in vitro by the A 0 and P proteins; (ii) is phosphorylated both in vitro and in vivo, probably as a result of an autophosphorylation reaction; and (iii) is active in an in vitro replication system (unpublished results) that is dependent on the A 0 and P proteins. METHODS AND MATERIALSStrains. The bacteria, bacteriophage, and plasmid strains used in this work have been described (2,9,10,14,15).Purification of dnaK' Protein. The details of the purification will be published elsewhere. Briefly, 12 g of wet paste of B178(pMOB45dnaK+) bacteria was lysed according to Shlomai and Kornberg (16). The supernatant was ...

Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.

“…2, the ATP-agarose affinity column is most effective in achieving the purification of the proteins, a result whose merit is obvious. It should also be pointed out that other laboratories have reported that the Escherichia coli DNA K protein, which by DNA sequence analysis has been shown to be 50% homologous to the Drosophila 70-kDa heat-shock protein (3), possesses both weak ATPase and autophosphorylating activities and appears to be involved in the replication of bacteriophage lambda DNA in infected cells (28,29). In the case of the mammalian 72-and 73-kDa proteins, we have tested the ability of the purified proteins to either hydrolyze ATP in vitro or to autophosphorylate.…”

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…Along with VirB4 and the CP (VirD4), VirB11 is thought to energize the type IV secretion machinery either for complex assembly, pilus production and/or substrate secretion. The NTPase activity of several VirB11-like proteins has been determined in vitro (Christie et al, 1989;Krause et al, 2000b;Rivas et al, 1997;Sexton et al, 2004), revealing a rather weak activity similar to the one observed for chaperons like DnaK (Zylicz et al, 1983). VirB11 of A. tumefaciens has been reported to additionally possess an autophosphorylation activity (Christie et al, 1989), however, such an activity was not detected for any other member of the family of VirB11-like proteins.…”

Heat Shock Proteins hsp60 and hsp70: Their Roles in Folding, Assembly and Membrane Translocation of Proteins

“…DnaK is an abundant, consitutively expressed protein with an apparent molecular weight of 70 000. The purified protein (ZYLICZ and GEORGOPOULOS 1984) possesses a weak ATPase activity (with a turnover number of about one ATP per minute) and can be autophosphorylated at threonine residues (ZYLICZ et al 1983). Its enzymatic activities are well studied (CEGIELSKA and GEORGOPOULOS 1989;DALIE et al 1990).…”

“…Its enzymatic activities are well studied (CEGIELSKA and GEORGOPOULOS 1989;DALIE et al 1990). The ATPase is DNA independent, but is modulated by AO and AP proteins in vitro and in vivo (ZYLICZ et al 1983). In contrast to ATP binding, ATP hydrolysis and the autophosphorylating activity depend on divalent cations (CEGIELSKA and GEORGOPOULOS 1989;DALIE et al 1990).…”