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Activity of the Hsp7o Chaperone Complex-Dnak, Dnaj, And Proc. Natl. Acad. Sci. USA Vol. 89, pp. 12108-12111, December 1992 Biochemistry Activity of the Hsp7O chaperone complex-DnaK, DnaJ, and GrpE- in initiating phage A DNA replication by sequestering and releasing A P protein (heat shock proteins/DNA replication/protein-protein interaction) HEIDI J. HOFFMANN, SUSAN K. LYMAN, CHI Lu, MARIE-AGNES PETIT, AND HARRISON ECHOLS Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720 Contributed by Harrison Echols, September 24, 1992 ABSTRACT Initiation of DNA replication by phage A occurs efficiently with less DnaK, and the unwinding reaction requires the ordered assembly and disassembly ofa specialized is more often bidirectional, indicating a more efficient disas- nucleoprotein structure at the origin of replication. In the sembly reaction (C. Wyman, C. Vasilikiotis, D. Ang, C. disasembly pathway, a set of Escherichia coli heat shock Georgopoulos, and H.E., unpublished work). Based on a proteins termed the Hsp7O complex-DnaK, DnaJ, and variety of data, the three-protein set of heat shock proteins GrpE-act with ATP to release A P protein from the nucleo- appears likely to function typically together. DnaJ and GrpE protein complex, freeing the DnaB helicase for its DNA- markedly stimulate the ATPase activity of DnaK, the bac- unwinding reaction. To investigate the mechanism of the terial homolog ofthe eukaryotic -70-kDa heat shock protein release reaction, we have exmned the interaction between P Hsp7O (14). Moreover, the three-protein set ("Hsp7O com- and the three heat shock proteins by glycerol gradient sedi- plex") acts together in other reactions, including control of mentation and gel electrophoresis. We have discovered an the heat shock regulator a32 (15, 16) and protein folding in ATP-dependent ternary interaction between P, DnaK, and vitro (17). DnaJ; this PIDnaKDnaJ complex is dissociated by GrpE. We In the work reported here, we have sought to understand have concluded that the function of the Hsp7O complex in the protein-protein interactions by which the Hsp7O complex sequestering and releasing P protein provides for the critical evicts P protein from the initiating nucleoprotein structure. step in the disassembly pathway. Based on our data and other We have identified an ATP-dependent interaction of P, work on protein folding, the formation of the P-DnaK DnaJ DnaK, and DnaJ that we believe to be the key intermediate complex might involve a conformational shift to a folding in releasing P; this ternary complex is turned over by GrpE intermediate of P. to free the individual proteins. We have concluded that DnaB is freed to become active as a helicase ("fired") by the Initiation ofDNA replication by phage A requires the ordered transient sequestering of P (possibly in a partially unfolded assembly and disassembly of a specialized nucleoprotein conformation). The activity of GrpE provides for a more structure (snup) at the viral origin of replication (oriA) (1, 2). efficient recycling action of DnaK and DnaJ. This sequential pathway serves to localize and "fire" the DnaB helicase of Escherichia coli, resulting in an origin- specific DNA-unwinding reaction that provides for the initi- MATERIALS AND METHODS ation of DNA replication (1, 2). The DnaB helicase is Proteins. 0 protein was purified by a modified version of localized at oriA by the phage 0 and P proteins. The 0 protein the procedure developed by Roberts and McMacken (18). P binds to four direct repeats in oriA DNA ("iterons"), each of protein (19), DnaJ (20), DnaK (21), and GrpE (22) were which is an inverted repeat (3, 4); the bound 0 forms a purified as described. All proteins were highly purified DNA-wound nucleoprotein structure, the "O-some," which (>90%o pure). IgG antibodies to these proteins were raised in serves as the scaffold for the rest ofthe assembly reaction (5). rabbits and purified over protein G affinity columns. RNase The P protein recruits DnaB by forming aP-DnaB complex (6, A was purchased from Sigma. 7), which binds in turn to the O-some (5, 8-11). The DnaB in Glycerol Gradient Sedimentation. Standard 100-II reaction the resultant O-P-DnaB snup is inert as a helicase (8-10), mixtures contained: 5 jug of P, 8 pug of DnaJ, 21 ,jg of GrpE, presumably because interaction with P quenches the enzy- and 56 ,ug of DnaK (where appropriate) in buffer R [40 mM matic activity of DnaB (6, 7). Hepes-KOH, pH 8.0/11 mM Mg(OAc)2/100 mM KCI]. In The intervention of bacterial heat shock proteins disas- standard P reactions, P was incubated with DnaJ or DnaK or sembles the oriA snup, freeing DnaB for its DNA-unwinding both for 5 min at 300C. ATP was then added, where appro- activity (8-12). The release of P is a characteristic feature of priate, to a final concentration of 2 mM, and the reaction the disassembly pathway and is likely to be the key biochem- mixture was incubated for another 5 min at 300C. GrpE was ical event for firing the helicase (9-12). Two disassembly then added, followed by a final incubation at 300C for 10 min. reactions have been characterized with pure proteins: Total incubation time for all reactions was 20 min at 30'C. In DnaK-DnaJ and DnaK-DnaJGrpE. In the more extensively denatured P reactions, P was incubated in 8 M urea at 520C studied DnaK*DnaJ pathway, the two heat shock proteins for 5 min and cooled to room temperature; the denatured P add to the O0P'DnaB snup, and an ATP-dependent step was then diluted 1:20 into solution containing one or more of initiates DNA-unwinding (11) and DNA replication (12, 13). the heat shock proteins, and incubations were carried out as The DnaK, DnaJ, and GrpE proteins act with reduced levels for native P. Reaction mixtures were then loaded onto ofDnaK to mediate the initiation ofDNA replication (10, 12). 10-30%o linear glycerol gradients in buffer R with 5 mM With the three heat shock proteins, DNA unwinding also dithiothreitol (preloaded with 5 ,.g of RNase A as an internal standard). Gradients for reactions with ATP also contained 2 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: Hsp7O, "70-kDa heat shock protein; snup, special- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ized nucleoprotein structure; oriA, phage A origin of replication. 12108 Downloaded by guest on September 27, 2021 Biochemistry: Hoffmann et al. Proc. Natd. Acad. Sci. USA 89 (1992) 12109 mM ATP. Centrifugation was carried out for 21 hr at 49,000 A 1)taK rpm with an SW6OTi rotor in a Beckman L65-5B ultracen- trifuge. Fractions of 110 ml were collected and analyzed on SDS/15% polyacrylamide gels (19%o for gradients containing both P and GrpE). Proteins were visualized by staining with Coomassie brilliant blue R250 (Bio-Rad). Native Agarose Gel e. Standard 5-Al reaction mixtures contained 50 ng ofP, 80 ng ofDnaJ, 200 ng ofGrpE, 1a.c ng were and 500 of DnaK in buffer R. Proteins mixed and N - incubated sequentially as in the sedimentation experiments. K zl,<CA. _0 Reaction mixtures were loaded onto 0.7% agarose gels in TGE (25 mM Tris base/190 mM glycine/1 mM EDTA) and ,w EWr * run at 100 V for 1.5 hr. Proteins then were denatured by [)3aIK1 -._ soaking the gel in TGS (25 mM Tris base/190 mM glycine/ 0.04%o SDS) for 30 min and transferred onto polyvinylidene fluoride membrane (Millipore) by using a Hoefer Transphor I)fldi -- electrophoresis unit. Proteins were detected by immunoblot- ting with the enhanced chemiluminescence (ECL) detection system (Amersham). -4 -i M. S RESULTS fRE~as.-\ "'.#'§ --iy :, Formation and Turnover of the PDnaK'DnaJ Complex. FiG. 1. Lack oftwo-protein interactions involving P. DnaK, and Sedimentation analysis. The release of P protein from the DnaJ in ATP. (A) A mixture ofP protein (5 ;Wg) and DnaK (56 1g) in initiating nucleoprotein structure requires DnaK and DnaJ buffer R with 2 mM ATP was incubated at 30'C for 20 min. The (8-11). To investigate the mechanism ofthis release reaction, reaction mixture was sedimented on a 10-301% glycerol gradient. we have examined the interactions between P, DnaK, and Fractions were collected, and proteins were fractionated by electro- DnaJ by velocity sedimentation in a glycerol gradient. The phoresis on 15% polyacrylamide gels. The figure shows the fractions proteins were mixed at stoichiometries typical of replication sequentially, starting with the top (most slowly sedimenting) fraction reactions, which utilize excess DnaK. The sedimentation at the far left. Only the top 19 fractions are shown of a total of 38 position of each protein was determined by polyacrylamide fractions. (B) DnaJ (8 ,ug) was incubated at 300C for20minwith DnaK gel electrophoresis of isolated fractions. Relative sedimen- (56 pg) in buffer R with 2 mM ATP. tation coefficients were calculated with respect to RNase A investigate the function of GrpE, we examined the influence as a marker protein. ofthis on the interactions revealed Because the disassembly reaction depends on ATP, we protein protein-protein by sedimentation analysis. When GrpE was added to P along were particularly interested in ATP-dependent interactions. with and ofP and In the presence of ATP, we found no evidence for any DnaK, DnaJ, ATP, the rapid sedimentation two-protein interactions sufficiently stable to persist in the DnaJ was not observed (Table 1). We have concluded that glycerol gradient. To show the sedimentation positions ofthe GrpE acts to turn over the P-DnaKDnaJ complex, freeing the individual proteins, an experiment is shown for a mixture of individual proteins.
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