From the
During the initiation of
Both genetic and biochemical studies indicate that bacteriophage
1.
2.
3. The concerted
action of three bacterial heat shock proteins: DnaK, DnaJ, and GrpE is
needed to activate an ori
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
Wild-type
phage
In this paper, we
characterize one mutant
During the early phase of
Our study of one mutant form of
The
A low affinity to DnaB results in reduced
capability of
Mutant DnaK756
protein was active in the replication reaction dependent on the
The ancestor of
modern bacteriophage
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
2.2 µg of DnaK, 50 ng of
DnaJ, 200 ng of GrpE, 65 ng of
2.5 µg of DnaK, 50 ng of DnaJ, 200 ng of GrpE, 65 ng of
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
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) .
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.
-
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) .
DNA template
(18) .
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.
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.
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
Reaction mixtures (100 µl) contained 25
mM Hepes-KOH, pH 7.6, 5 mM MgClP-DnaB Complex by Gel Filtration
Chromatography
, 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.
Cloning and Overexpression of
Plasmid pIK12 was constructed by placing a P (
A66)
Gene
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.
Replication activity of purified Protein Retains Replication Activity in Vitro,
in Both a Crude Enzyme System and Reaction Reconstituted with Purified
Proteins
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.''
To further
characterize the mutant Protein Binds to DnaB Helicase with a Low
Affinity and the Complex Formed Is Unstable
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.
The Protein Has a Weak Inhibitory Effect on oriC
Plasmid Replication in Vitro
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
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 P Gene
A66 Needs a Longer Development
Time
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.
The
molecular mechanism of -dependent, in Vitro Replication of
dv
Plasmid Is Active at Low Concentrations of DnaK Protein
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. Either
P 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
One of the phenotypic
characteristics of phages harboring -dependent
in Vitro Replication of
dv Plasmid
-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.
The simplest -dependent Replication of M13 ssDNA
Reconstituted with Purified Proteins Is Active in the Absence of Heat
Shock Proteins
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.
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.
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
The product of the dnaB gene is the only
helicase active in both replication of E. coli chromosome and
bacteriophage Protein with DnaB
Helicase
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) .
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.
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
Once Protein Prepriming
Complex
P protein forms the
P-DnaB complex, it
immediately binds to the ori
DNA-
O initiation
complex, which results in the assembly of an
ori
DNA-
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 ori
DNA-
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 ori
DNA 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
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 Preprimosome Role of Heat
Shock Proteins
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.
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 dnaK
and 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
DNA replication
activity dependent on the presence of -dependent Replication of M13 ssDNA
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
In our opinion, understanding the biochemical
mechanisms behind the phenotype of the mutation DNA Replication and Role of Heat Shock
Proteins
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.
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
, 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
P, 100 ng of
were added
to the reaction mixture as indicated.
Table:
Heat shock proteins
are dispensable in -dependent replication of M13 ssDNA
P,
and 130 ng of
were added to the reaction mixtures as
indicated.
O,
O protein;
P,
P protein;
,
P protein with mutation (
A66); dsDNA,
double-stranded DNA; ssDNA, single-stranded DNA; hsp, heat shock
proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.