©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Requirement for Molecular Chaperones in DNA Replication Is Reduced by the Mutation in P Gene, Which Weakens the Interaction between P Protein and DnaB Helicase (*)

Igor Konieczny (§) , Jaroslaw Marszalek (¶)

From the (1) Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

During the initiation of DNA replication, the host DnaB helicase is complexed with phage P protein in order to be properly positioned near the ori-O initiation complex. However, the P-DnaB interaction inhibits the activities of DnaB. Thus, the concerted action of bacterial heat shock proteins, DnaK, DnaJ, and GrpE, is required to activate the helicase. Wild-type phage cannot grow on the E. coli dnaB, dnaK, dnaJ, and grpE mutants. However, phage with a mutation in the P gene, is able to produce progeny in these mutants as well as in the wild-type bacteria. Purified mutant protein reveals a much lower affinity to DnaB than wild-type P, and the -DnaB complex is unstable. Also, a very low concentration of DnaK protein is sufficient to activate the helicase in a replication system based on dv dsDNA. In that system, the mutant DnaK756 protein, inactive in the P-dependent replication, revealed its activity in the -dependent reaction. The O-P-dependent replication system based on M13 ssDNA efficiently replicates DNA in the absence of any chaperone protein, unless P is substituted by the mutant protein. Data presented in this paper explain why phage is able to grow on wild-type and dnaK756 bacteria.


INTRODUCTION

Both genetic and biochemical studies indicate that bacteriophage has evolved an efficient strategy for recruiting bacterial enzymatic machinery to replicate its own genome. Only two phage proteins, the products of O() and P genes, are involved in the replication of DNA.

1. O initiator protein specifically recognizes and binds four iterons located within the origin sequence ( ori) (1) . Several O molecules form a nucleoprotein complex called the O-some and alter the DNA topology near the ori sequence, resulting in the formation of an active initiation complex (2, 3, 4) .

2. P protein performs a central role in bringing host proteins to replicate the viral DNA. P forms a tight complex with the bacterial DnaB helicase (5, 6) and directs the helicase for binding to the ori initiation complex. To perform this function, P protein has to compete with the host DnaC protein that also forms a complex with DnaB helicase and fulfills the analogous role in bacterial replication (7) . Biochemical experiments indicate that P protein has a higher affinity to DnaB than the DnaC protein does and efficiently sequesters DnaB helicase from the host replication pathway (7) . The P-DnaB protein complex interacts with the ori-O nucleoprotein complex and an ori-O-P-DnaB ternary complex is formed through specific protein-protein interactions between P and O proteins (8, 9) . The ori-O-P-DnaB complex is stable, as it can be isolated by gel permeation chromatography and subsequently demonstrated to be active in DNA replication. (10, 11, 12, 13) . The strong P-DnaB interaction inhibits the helicase activity of DnaB protein (5, 14) . Thus, subsequent steps are needed to liberate the helicase from the complex with P protein.

3. The concerted action of three bacterial heat shock proteins: DnaK, DnaJ, and GrpE is needed to activate an ori-O-P-DnaB complex (12, 15) . The key player in this process is DnaK protein (hsp70 homolog), which in an ATP-dependent reaction catalyzes translocation of P protein within the preprimosomal complex in such a way that it is no longer an inhibitor of DnaB helicase (13) . DnaJ and GrpE assist in this reaction by increasing the specificity of the DnaK-P interaction (16, 17) .

4. Once DnaB is established as a processive helicase, those additional host proteins that are required to propagate a replication fork and synthesize daughter DNA strands, spontaneously interact with both DnaB protein and the DNA template (18) .

Wild-type phage cannot grow on some of the dnaB, dnaK, dnaJ, or grpE mutants of Escherichia coli. However, phages harboring mutations, called , in the P gene, are able to produce progeny in these bacterial mutants, as well as in the wild-type bacteria (19, 20) . As many as 14 different -type missense mutations were isolated and sequenced, all of them located in the C terminus of the P gene (21) . These genetic observations suggest that the interactions of gene product with DnaB, DnaK, DnaJ, and GrpE proteins are different from the interactions of wild-type P protein.

In this paper, we characterize one mutant P protein (hereafter cited as the ), which contains a leucine in place of an arginine at position 137 (mutation A66) (21) . Phage A66 is able to produce progeny in wild-type bacteria as well as in bacterial mutants: dnaB (groPA15), dnaK756 (19, 21) , grpE280, and dnaJ259.() Our results at least partially explain why phage is able to grow on these bacterial mutants and also shed a new light on the biochemical mechanism of initiation of DNA replication and the role of heat shock proteins in this process.


MATERIALS AND METHODS

Proteins

Highly purified replication proteins were (90% or greater purity) used. The DnaC, DNA polymerase III holoenzyme, and SSB proteins were as described (22) . The O protein was purified according to Ref. 23, protein according to Ref. 24, and DnaB protein according to Ref. 25. The C-labeled protein (13,100 cpm/µg of protein) was purified according to Ref. 10. The DnaA, GyrA, GyrB, DnaG, and HU proteins and anti-DnaC rabbit serum were the kind gifts of Dr. Jon M. Kaguni (Michigan State University). The P, DnaK, DnaK756, DnaJ, and GrpE proteins were the kind gifts of Dr. Krzysztof Liberek (University of Gdansk). The C-labeled P (38,000 cpm/µg of protein) and anti-P rabbit serum were the kind gifts of Dr. Maciej Zylicz (University of Gdansk). The anti- rabbit serum was the kind gift of Dr. Grzegorz Wegrzyn (University of Gdansk).

Bacteria, Bacteriophages, and Plasmids

E. coli strains: MM294, supE44, hsdR, endA1, pro, thi 1 (26) ; MM294 harboring plasmids, pIK12, pGP1-2 (this work); R594, galK2, galT22, rpsL179, lac (26) ; WM433, leu19, pro19, trp25, his47, thyA59, arg28, met55, deoB23, lac11, gal11, str56, sul1, hsdS, dnaA204 (27) . Bacteriophages: Pwt (cIb2) (from Dr. A. Klein, University of Heidelberg); A66 (cIb2 A66) (28) . Plasmids: pRLM4 dv, P, cro ts, c II, O, P, Tn5 (kan) (29) ; pGW2 dv, P, cro, c II, O, A66, Tn5 (kan) (30) ; M13 oriC2LB5 contains oriC sequence (31) ; M13mp18 (26) ; pGP1-2 harboring T7 RNA polymerase gene (32) ; pBluescript II SK harboring 10 promoter, used as a vector (Stratagene); pIK12 (this work).

Replication Reactions Dependent on Crude Protein Fraction

Crude bacterial extract (FII) was prepared from E. coli strain WM433 according to the procedure described in Ref. 33. Unless otherwise noted, reaction mixtures (25 µl) for dv plasmid replication contained: 400 ng of pRLM4, 100 ng of O, and P or proteins as indicated and were assembled as described (33) at 0 °C, then incubated at 30 °C for 25 min, to measure DNA synthesis as described (29) . For oriC replication, 200 ng of M13 oriC2LB5 and 100 ng of DnaA protein were added to the reaction mixture.

Replication Reactions Reconstituted with Purified Proteins

Reaction mixtures (25 µl) for dv plasmid replication were assembled as described (12) and supplemented with 400 ng of pRLM4, 50 ng of DnaJ, 200 ng of GrpE, 2.2 µg of DnaK, 140 ng of O, and P or as indicated. For replication of ssDNA template (34) , reactions were similar to those for dsDNA except for the following: dv was replaced by 480 ng of M13mp18 ssDNA; the ATP regeneration system, GyrA/B, and HU proteins were omitted. For oriC replication (22) , reaction mixtures were supplemented with 200 ng of M13 oriC2LB5, 100 ng of DnaA, and 56 ng of DnaC. Reactions were assembled on ice and incubated for 30 min at 30 °C.

Isolation of P-DnaB Complex by Gel Filtration Chromatography

Reaction mixtures (100 µl) contained 25 mM Hepes-KOH, pH 7.6, 5 mM MgCl, 200 mM KCl, 2 mM dithiothreitol, 0.5 mM ATP, 10% (v/v) glycerol, and DnaB, P, and proteins as indicated. After incubation for 10 min at 30 °C, reaction mixtures were chromatographed through a Bio-Gel A-0.5m column (0.5 8 cm) equilibrated at room temperature in the same buffer as above, but without glycerol. Fractions (100 µl) were collected at room temperature.


RESULTS

Cloning and Overexpression of P (A66) Gene

Plasmid pIK12 was constructed by placing a DNA restriction fragment HincII (38563)- SstII (40389), which contains the O gene and the P gene with a A66 mutation, into pBluescript II SK vector. The DNA fragment is derived from dv plasmid pGW2 (30) . For overexpression of P (A66), the T7 RNA polymerase system with plasmid pGP1-2 (32) and plasmid pIK12 were used.

Protein Retains Replication Activity in Vitro, in Both a Crude Enzyme System and Reaction Reconstituted with Purified Proteins

Replication activity of purified protein was tested in a reaction dependent on a crude extract of E. coli strain WM433 harboring a mutation in the dnaA gene. Such a crude enzyme system supported the replication of oriC plasmids when supplemented with purified DnaA protein and was active in replication of dv plasmid when purified O and P proteins were added instead of DnaA protein. To measure DNA replication, increasing amounts of P or proteins were added into reaction mixtures containing a fixed level of dv DNA, O protein, and the bacterial extract (Fig. 1). The protein was active in this replication system, but the maximal rate of DNA synthesis was observed at 10-fold higher concentrations of (600 ng/reaction) compared with P (65 ng/reaction) (Fig. 1 A). We were concerned that the low specific activity of might have been due to a partial inactivation of the mutant protein during the purification procedure. However, this possibility was highly unlikely, because in the replication reaction reconstituted with purified proteins, exhibited maximal activity at 100 ng/assay, when the optimal level of wild-type P was 65 ng/reaction (Fig. 1 B). Thus, both mutant and wild-type proteins have a similar specific activity. In the crude enzyme system, replication proteins are supplemented in the form of the bacterial extract and contained DnaB and DnaC proteins. It was previously established that a stable DnaB-DnaC complex is formed both in vivo and in vitro (35, 36, 37) . Thus, one of the explanations of our results is that the high concentration is needed in the crude enzyme replication system, because the affinity of protein to DnaB helicase might be low. Therefore, this protein, in contrast to P, could not successfully compete with the host DnaC protein for binding to DnaB helicase. In order to test this hypothesis, the standard crude extract replication reaction was divided into two stages (). First, protein was incubated with the purified DnaB protein to preform the -DnaB complex, and, in the second stage, replication components missing in the first stage (including crude bacterial extract) were added, and replication activity was subsequently measured. Preincubation of with DnaB helicase before the initiation of DNA replication resulted in a 10-fold decrease of the optimal protein concentration required for DNA synthesis (). Moreover, this concentration was almost the same as the optimal level required in the reaction reconstituted with purified proteins (Fig. 1 B). In a control experiment, preincubation of wild-type P protein with DnaB helicase had no effect on the specific replication activity of P protein (). These results further support our hypothesis, that both mutant and wild-type proteins have a similar specific replication activity but differed significantly in their affinity to bind to DnaB helicase.


Figure 1: Replication activity of protein. dv plasmid replication reactions in a crude enzyme system ( A) or in a system reconstituted with purified proteins ( B) were assembled as described under ``Materials and Methods.''



Protein Binds to DnaB Helicase with a Low Affinity and the Complex Formed Is Unstable

To further characterize the mutant protein, the formation of -DnaB complex and the stability of that complex were tested. In the presence of DnaB protein, after gel filtration chromatography, 61% of C-labeled P protein was detected in the form of a P-DnaB complex (Fig. 2 A). In contrast, under the same experimental conditions, only 34% of protein was detected in a complex with DnaB (Fig. 2 B). A more dramatic difference was observed in the presence of a nonionic detergent, Triton X-100 (0.02% v/v final concentration). The amount of P bound to DnaB remained the same, but only 3% of was complexed with DnaB (results not shown). Subsequently, the stability of the P-DnaB complex was directly tested by chasing radiolabeled [C]P bound to DnaB with an excess of nonradioactive P. The C-labeled P protein was first preincubated with DnaB protein to ensure P-DnaB complex formation, then a 20-fold molar excess of nonradioactive P protein was added and the reaction mixture was chromatographed through the gel filtration column. Under these conditions, over 42% of radioactive P remained bound to DnaB (Fig. 2 A). Thus, once formed, the P-DnaB complex does not dissociate easily. In contrast, under similar experimental conditions, the complex of with DnaB helicase is very unstable. A 20-fold molar excess of nonradioactive protein reduced the binding of [C] from 34% to 2% (Fig. 2 B). Competition between wild-type P and mutant proteins for binding to DnaB was also tested (Fig. 3). In the control experiment, radioactive [C]P protein was mixed with a 20-fold molar excess of nonradioactive P before incubation with DnaB. In that situation, only 3% of the radioactive protein was detected in the complex with the helicase (Fig. 3). However, when [C]P was mixed with a 20-fold excess of nonradioactive protein, and then incubated with DnaB, as much as 44% of the radioactivity was detected in the complex with DnaB (Fig. 3). These results indicate that the affinity of protein to DnaB is low and it cannot compete successfully with the wild-type counterpart.


Figure 2: Formation and stability of P-DnaB and -DnaB complexes. A, [C]P protein (250 ng) was incubated with DnaB protein (750 ng) for 10 min at 30 °C in the reaction conditions described under ``Materials and Methods.'' The reaction mixture (100 µl) was either applied directly to a gel filtration column () or a 20-fold excess of nonradioactive P protein was added to the reaction (), and incubation was continued for 10 min at 30 °C, followed by column chromatography. , [C]P (250 ng) alone, applied directly to a gel filtration column. 100-µl fractions were collected and the radioactivity was estimated in a scintillation counter. The position of the P-DnaB complex (indicated by arrow) was determined by Western blot analysis using anti-P and anti-DnaB serum, following SDS-polyacrylamide gel electrophoresis separation of aliquots of fractions collected during chromatography (results not shown). B, [C] experiment was performed exactly as in A except that a 20-fold excess of non-radioactive was used for chasing. , [C] + DnaB; , [C] + DnaB + nonradioactive ; , [C] alone. The position of the -DnaB complex is indicated by the arrow.




Figure 3: Competition between P and proteins for binding to DnaB helicase. [C]P protein (350 ng) was mixed with a 20-fold excess of either nonradioactive P protein () or with a 20-fold excess of nonradioactive protein (), then DnaB protein (700 ng) was added to the reaction mixtures (100 µl), which were incubated for 10 min at 30 °C, followed by size chromatography. Further treatment was as described in the legend to Fig. 2. The arrow indicates the position of the P-DnaB complex.



Protein Has a Weak Inhibitory Effect on oriC Plasmid Replication in Vitro

The P protein exhibits a very strong inhibitory effect on the oriC plasmid replication in a crude enzyme system. This is due to the sequestering of the DnaB helicase from the DnaB-DnaC complex resulting in the formation of an inert P-DnaB complex (7) . We have also observed this phenomenon (Fig. 4 A) and even stronger inhibition of oriC replication in reactions reconstituted with purified proteins (Fig. 4 B). At an equimolar ratio of DnaC to P, replication reactions reconstituted with purified enzymes was inhibited by over 50%. In contrast, mutant protein exhibited a very weak, if any, inhibitory effect on oriC replication in both the crude enzyme system and the reaction reconstituted with purified proteins (Fig. 4). Competition between DnaC and P or proteins for binding to DnaB helicase was determined directly in the following experiment. To preform DnaB-DnaC complex, DnaB protein was incubated with DnaC (1:4 molar ratio), then P or protein was added (2-fold in excess over DnaC), and incubation was continued for another 10 min. Following separation of proteins by sizing chromatography, P and a reduced amount of DnaC protein were detected bound to DnaB (Fig. 5). Formation of a similar mixed (DnaB-DnaC-P) complex was previously reported (7) . In contrast, no was observed in the fraction containing both DnaB and DnaC proteins. Instead, bands corresponding to are well visible at the position of free protein. Thus protein, at relatively low concentrations, is unable to sequester DnaB from the DnaB-DnaC complex.


Figure 4: Inhibition of oriC replication by P and proteins. oriC plasmid replication reactions in a crude enzyme system ( A) or a system reconstituted with purified proteins ( B) were assembled on ice as described under ``Materials and Methods,'' except that DnaA protein was omitted, and P or proteins were added at the indicated amounts, then mixtures were incubated for 10 min at 30 °C. Following this, replication reactions were started by the addition of DnaA protein and incubated for 30 min at 30 °C.




Figure 5: Competition between or P and DnaC proteins for binding to DnaB protein. DnaC protein (600 ng) was incubated with DnaB protein (250 ng) for 10 min at 30 °C, then reaction mixtures (100 µl) containing 25 mM HEPES-KOH, pH 7.6, 5 mM MgCl, 100 mM KCl, 2 mM dithiothreitol, 0.5 mM ATP, 10% (v/v) glycerol, and 0.1 mg/ml bovine serum albumin were treated in the following way: applied directly on the chromatographic column ( C), P protein (1300 ng) was added ( A), or protein (1300 ng) was added ( B) and reactions were incubated for another 10 min at 30 °C. Following chromatographic separation, fractions were collected and electrophoresed on SDS-polyacrylamide gel electrophoresis. After electrotransfer on nitrocellulose membrane, DnaB, DnaC, P, and proteins were detected by immunoblot analysis with rabbit antisera specific for these proteins. Colorimetric detection of the bound rabbit antibodies was by use of biotinylated goat anti-rabbit IgG and streptavidin-alkaline phosphatase conjugate.



One-step Growth Experiment Indicates That Phage Harboring a Mutation in P Gene A66 Needs a Longer Development Time

The intracellular concentration of phage progeny was measured at different time intervals after phage infection using a one-step growth technique. One hour after infection, an average bacterial cell contained about 20 wild-type phage particles (Fig. 6). In contrast, bacteria infected by mutant A66 phage contained the same amount of progeny 20 min later. Thus, the mutant phage needs longer development time in vivo.


Figure 6: Phage harboring mutation (A66) in P gene, revealed a 20-min lag period in one-step growth experiment. A one-step growth experiment (49) was performed at 30 °C in E. coli R594 strain with a multiplicity of infection of 0.05 of phage Pwt and phage A66. The intracellular phage progeny were estimated by plating on the indicator strain (R594) at 30 °C. pfu, plaque-forming unit.



-dependent, in Vitro Replication of dv Plasmid Is Active at Low Concentrations of DnaK Protein

The molecular mechanism of dv plasmid replication was studied extensively in vitro in reactions reconstituted with purified proteins (11, 12, 15, 34, 38) . In addition to phage O and P proteins, other host protein factors, including three host heat shock proteins DnaK, DnaJ, and GrpE, have to be added to the in vitro ori DNA replication assay. Knowing that phage produce progeny in dnaK, dnaJ, and grpE mutants of E. coli, we tested the influence of those heat shock proteins on dv plasmid replication dependent on protein. All three heat shock proteins were required to replicate dv plasmids in both P- and -dependent reactions (). However, we have found that -dependent replication reactions were fairly active at very low concentrations of DnaK protein. Under these same conditions of low DnaK concentrations, P-dependent replication activity was not detectable (Fig. 7).


Figure 7: dv plasmid replication reaction dependent on protein is active at very low concentrations of DnaK protein. EitherP or proteins were added to the replication reaction mixtures, reconstituted with purified enzymes according to the procedure described under ``Materials and Methods,'' except that indicated amounts of DnaK protein were added. 100% refers to the maximal replication activity (determined in the presence of 2.2 µg of DnaK): 280 pmol/30 min for P and 243 pmol/30 min for -dependent systems.



Mutant DnaK 756 Protein Is Active in -dependent in Vitro Replication of dv Plasmid

One of the phenotypic characteristics of phages harboring -type mutations is growth in bacterial strains harboring mutations in hsp genes. One of these E. coli mutants is dnaK756 (19, 21) . In such a strain, wild-type phage cannot produce progeny. In agreement with this, purified DnaK756 protein does not support in vitro replication of dv plasmid dependent on wild-type P protein (Fig. 8 A). However, DnaK756 protein was active in replication reactions reconstituted with purified proteins, when P was substituted with the mutant protein (Fig. 8 A). The kinetics of -dependent replication reactions in the presence of either wild-type DnaK or mutant DnaK756 proteins was very similar. However, in the presence of DnaK756, the rate of DNA synthesis was about 3-fold lower (Fig. 8 B). These results suggest that because of the low stability of the -DnaB complex, DnaK756 mutant protein was active enough to trigger a partial rearrangement of the prepriming complex leading to DNA replication.


Figure 8: DnaK756 protein is active in dv plasmid replication reaction dependent on protein. A, either P or were added to the replication reaction mixtures reconstituted with purified proteins as described under ``Materials and Methods,'' except that DnaK protein was missed and indicated amounts of DnaK756 were added. 100% refers to the maximal replication activity (determined in the presence of 2.2 µg of DnaK): 300 pmol/30 min for P- and 186 pmol/30 min for -dependent reactions. B, either DnaK (2.2 µg) () or DnaK756 (2.4 µg) () proteins were added to the standard replication reactions reconstituted with purified enzymes in the presence of (68 ng) protein.



-dependent Replication of M13 ssDNA Reconstituted with Purified Proteins Is Active in the Absence of Heat Shock Proteins

The simplest replication system reconstituted with purified enzymes uses M13 ssDNA as a template (34) . This reaction is not specific to the ori sequence, but, at high concentrations of SSB protein, the reaction is completely dependent on the presence of O and P. The presence of DnaK, DnaJ, and GrpE proteins in the reaction mixture is obligatory for replication activity in a P-dependent reaction (I). However, in the absence of any hsp proteins, 70% of maximal -dependent replication activity was observed. Moreover, the presence of a single heat shock protein or any combination of two hsp proteins did not alter the rate of DNA synthesis. Only in the presence of all three heat shock proteins was the replication activity increased from 175 pmol/30 min to 280 pmol/30 min (I). This result suggests that, in the case of the prepriming complex formed on ssDNA, the activation of DnaB protein occurred spontaneously, without the action of hsp proteins. Probably, this prepriming complex is much less stable than its counterpart formed at the ori region of dsDNA.


DISCUSSION

During the early phase of phage development, P protein plays a key role in switching the host replication machinery to replicate DNA. The molecular mechanism of P protein action can be divided into three separate reactions: (i) formation of a P-DnaB complex; (ii) binding of P-DnaB complex with the O-some complex, resulting in the formation of a prepriming complex; (iii) rearrangement of the prepriming complex and dissociation of DnaB helicase from P-dependent inhibition, by the action of three hsp proteins: DnaK, DnaJ, and GrpE.

Our study of one mutant form of P protein (A66) indicates that a single substitution of leucine by arginine at position 137 alters the biochemical properties of P protein and affects the first and the third of the above listed reactions.

Interaction between Protein with DnaB Helicase

The product of the dnaB gene is the only helicase active in both replication of E. coli chromosome and bacteriophage DNA (14, 39) . DnaB protein is active as an hexamer, which forms a complex with 6 molecules of ATP. Physical interaction between P and DnaB proteins was predicted based on genetic experiments several years ago (19, 20, 40) and later confirmed in vitro (5, 6, 7) . The DnaB-P complex is stable, as it was isolated using several biochemical techniques: ion exchange chromatography (6) , glycerol gradient centrifugation (7) , and size chromatography (this paper). The striking stability of the DnaB-P complex was confirmed directly in this paper (Fig. 2 A). Formation of a P-DnaB complex alters the biochemical properties of both components; P bound to DnaB became insensitive to inhibition by N-ethylmaleimide (5) , on the other hand, binding of P inhibits the ATPase activity of DnaB (5, 6) which appears to correlate with its function as a helicase (14, 39) . DnaB complexed with P cannot efficiently interact with primase or forms of the active primosome, as the dnaB-P complex is inert in both general priming reactions (7) and in the conversion of X174 ssDNA into dsDNA (5, 7) .

The A66 phage mutant was selected on E. coli groPA15 strain harboring a mutation in the dnaB allele (28) . Wild-type phage does not produce progeny (19) in this genetic background. Thus, one could expected an altered interaction between mutant protein and the DnaB helicase. We have found that protein has a much lower affinity to DnaB helicase then P, and the complex which forms is unstable. This conclusion is based on the following evidence: (i) after the isolation of a DnaB- complex by gel filtration chromatography, a substantial fraction of protein (66%) remained unbound to DnaB, (ii) in the presence of low concentrations of a nonionic detergent, Triton X-100 (which has no effect on P-DnaB interaction), the -DnaB complex is not stable, (iii) 20-fold excess of protein over the wild-type P protein is not sufficient to outcompete the latter from a complex with DnaB, (iv) the complex formed in the presence of is unstable, as a 20-fold excess of unlabeled protein displaces [C] from preformed -DnaB complex.

A low affinity to DnaB results in reduced capability of protein to sequester the helicase from the DnaC-DnaB complex. This feature may have significant influence on the development of bacteriophage harboring a mutant gene. A one-step growth experiment indicated that mutant phage produced progeny with an eclipse period 20 min longer than that for the wild-type counterpart. Consistent with this, a 10-min lag in DNA synthesis of phage A66 was observed (28) . On the other hand, dv plasmids with a A66 mutation are characterized by a 10-fold lower copy number.() We would like to interpret these results according to our findings in vitro. After infection, mutant phage synthesize protein, which at low concentrations is unable to sequester DnaB helicase from the pathway of host chromosome replication. Thus, some additional time is needed to increase the intracellular level of protein. Alternatively, the delay in phage growth could be ascribed to a very poor efficiency of assembly of a preprimosome complex when the mutation is present.

Formation of Protein Prepriming Complex

Once P protein forms the P-DnaB complex, it immediately binds to the oriDNA-O initiation complex, which results in the assembly of an oriDNA-O-P-DnaB prepriming complex. This complex is formed by means of protein-protein and protein-DNA interactions and is very stable (9, 10, 11, 13, 38) . It was possible that, in the presence of the oriDNA-O initiation complex, protein could bind more tightly to DnaB, as the formation of preprimosome could stabilize the interaction between these two proteins. We tested this prediction and have found that oriDNA and O protein have only a small effect on the fraction of protein which entered the -DnaB complex (34-46%) (results not shown). Thus, formation of a prepriming complex does not affect protein affinity to DnaB helicase.

Activation of the Preprimosome Role of Heat Shock Proteins

To trigger the helicase activity of DnaB protein, the nucleoprotein complex has to be partially disassembled by three host hsp proteins (DnaK, DnaJ, and GrpE). The molecular mechanisms of this process was studied in vitro in replication systems reconstituted with the purified proteins (10, 12, 13, 15, 34, 41) . When protein was substituted for P in replication of dv plasmid, activity was completely dependent on the presence of all three hsp proteins. However, a very low concentration of DnaK protein was sufficient to observe DNA synthesis. It was shown previously that the concentration of DnaK protein is important for the efficient replication of dv plasmid in vitro (12, 13, 42) . The replication reaction dependent on just two heat shock proteins (DnaJ and DnaK) needs an order of magnitude higher concentration of DnaK protein, compared with all other replication proteins (10, 15) . However, the addition of GrpE protein reduces the concentration of DnaK in the reaction mixture 5-fold (12, 15) . In this paper, we were using the three heat shock protein system and observed that DnaK concentrations could be reduced even further, when P protein was replaced by protein. Moreover, concentrations of DnaJ and GrpE proteins were standardized for optimal activity of P dependent replication. Therefore, it is possible that both DnaJ and GrpE could also be active at much lower concentrations in the replication reaction dependent on protein.

Mutant DnaK756 protein was active in the replication reaction dependent on the protein; however, in the presence of DnaK756, the rate of DNA synthesis was 3-fold lower compared with the wild-type DnaK. Mutant DnaK756 protein, as it was observed before (12, 43) , was completely inert in replication reactions dependent on wild-type P. This protein is also unable to form a stable complex with P protein (41) or to dissociate P from the preprimosome complex in vitro (10) . On the other hand, DnaK756 protein was partially active in complex formation with bovine pancreatic trypsin inhibitor (44) and the mutant form of alkaline phosphatase- phoA61 (45) ; it also forms the DnaK756--DnaJ ternary complex.() Our results may explain why phage is able to grow on wild-type dnaKand dnaK756 bacteria (19, 21) . Probably, due to the low affinity of protein to DnaB helicase, the prepriming complex formed in the presence of is unstable. Thus, less ``chaperone power'' is sufficient to free DnaB from -dependent inhibition.

Heat Shock Proteins Are Dispensable in -dependent Replication of M13 ssDNA

DNA replication activity dependent on the presence of O and P proteins can be measured by using, as a template, either dv plasmid (29) or circular ssDNA (34) . The latter does not contain ori sequences, and the preprimosomal (O-P-DnaB) complex is assembled at a random position on ssDNA. This complex is stable and it also needs action of three hsp proteins for activation (34) (I). However, the function of DnaB protein in the replication of ssDNA is different from that in the replication of dsDNA template. The helicase activity is not obligatory, but the interaction between DnaB protein and primase (DnaG) is critical, resulting in the formation of the primosome, and finally primer synthesis. We have found that activation of the preprimosome formed on ssDNA in the presence of protein occurs spontaneously, without participation of hsp proteins. This is very similar to the ABC priming reaction (46) , in which three E. coli proteins: DnaA, DnaB, and DnaC form a preprimosome on ssDNA template. In that replication system, DNA synthesis starts after the spontaneous dissociation of DnaC protein (22, 47) . By analogy, unstable associations between and DnaB proteins can dissociate spontaneously after formation of a prepriming complex. The DNA structure may also play an important role in the stability of preprimosome. In oriC-dependent replication of dsDNA, in contrast to ABC priming, DnaC protein remained bound to the template after activation of preprimosome (47) . Analogously, the protein-protein and protein-DNA interactions within the preprimosome assembled at the ori sequence can be more stable compared with the preprimosome assembled on M13 ssDNA. Therefore, hsp proteins are needed only for the activation of a nucleoprotein complex formed on dsDNA.

Evolution of DNA Replication and Role of Heat Shock Proteins

In our opinion, understanding the biochemical mechanisms behind the phenotype of the mutation A66 in P gene sheds a new light on the evolution of bacteriophage replication and the role of heat shock proteins in this process. As many as 14 missense mutations exhibiting the -type phenotype were identified at the C-terminal domain of P gene (21) . All of them have a common phenotype, as the mutant phage grow in bacteria harboring ts mutations in dnaK, dnaJ, and grpE genes, as well as in the wild-type bacteria. Assuming that each P protein with a mutation is characterized by a low affinity to DnaB helicase, one can say that it is relatively easy by selection to change wild-type P protein into its counterpart.

The ancestor of modern bacteriophage evolved a biochemical mechanism, allowing it to recruit bacterial enzymes for replication of DNA, which uses principles different from the one developed by phage P1 or different plasmids pSC101, F, R6K, RK2, R1, which also replicate in the same host. Instead of using the bacterial DnaA, DnaB, and DnaC prepriming system (18) , phage encodes its own initiation O protein and P protein (an analog of host DnaC) to direct DnaB helicase into the prepriming complex. Probably, ancestors of P protein did not compete for DnaB helicase as efficiently as modern P does. There were probably more like protein; therefore, hsp proteins were not needed for activation of the prepriming complex. Selection pressure for fast growth and development of phage, produced modern P protein, which is more efficient in sequestering DnaB helicase, but the side effect of this adaptation is the great stability of the preprimosome. Thus, heat shock proteins, whose intracellular levels were already elevated as the result of phage infection (48) , became involved in the activation of the prepriming complex. According to this hypothetical scenario, the well established function of heat shock proteins in DNA replication would be a unique and relatively recent adaptation to the specific mode of replication use by this parasite.

  
Table: Preformed -DnaB complex is active in a crude enzyme system of dv DNA replication at low concentration of protein

A standard crude enzyme replication reaction (see ``Materials and Methods'') was divided into two stages. First, a preincubation mixture (10 µl) contained: 40 mM Hepes-KOH, pH 8.0, 7% PVA, 2 mM ATP, 11 mM Mg, DnaB (81 ng), and P or as indicated, was incubated for 5 min at 30 °C to form P- or -DnaB complexes. Second, other replication reaction components (including the crude enzyme extract), were added to the preincubation mixture (to a final reaction volume of 25 µl), and DNA synthesis was determined after a 25-min incubation at 30 °C.


  
Table: dv plasmid DNA replication reconstituted with purified proteins

2.2 µg of DnaK, 50 ng of DnaJ, 200 ng of GrpE, 65 ng of P, 100 ng of were added to the reaction mixture as indicated.


  
Table: Heat shock proteins are dispensable in -dependent replication of M13 ssDNA

2.5 µg of DnaK, 50 ng of DnaJ, 200 ng of GrpE, 65 ng of P, and 130 ng of were added to the reaction mixtures as indicated.



FOOTNOTES

*
This research was supported in part by the State Committee for Scientific Research project 6P2030 42 06 and by grant from the United States-Poland Maria Sklodowska-Curie Joint Fund II (MEN/NSF-90-30). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the State Committee for Scientific Research Project 4 0045 91 01.

To whom correspondence and reprint requests should be addressed. Tel.: 48-58-310-072; Fax: 48-58-310-072; E-mail: marszalk@biotech.univ.gda.pl.

The abbreviations used are: O, O protein; P, P protein; , P protein with mutation (A66); dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; hsp, heat shock proteins.

I. Konieczny, G. Wegrzyn, and K. Taylor, unpublished results.

G. Wegrzyn and K. Taylor, personal communication.

K. Liberek, personal communication.


ACKNOWLEDGEMENTS

We thank Karol Taylor, Maciej Zylicz, and Donald Helinski for continuous interest and generous support throughout this work. We thank Krzysztof Liberek for helpful discussions and Ted Hupp and Jon M. Kaguni for critical reading of the manuscript.


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