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(Received for publication, January 18, 1996) From the
After T4 bacteriophage infection of Escherichia coli,
the enzymes of deoxyribonucleoside triphosphate biosynthesis form a
multienzyme complex that we call T4 deoxyribonucleoside triphosphate
(dNTP) synthetase. At least eight phage-coded enzymes and two enzymes
of host origin are found in this 1.5-mDa complex. The complex may
shuttle dNTPs to DNA replication sites, because replication draws from
small pools, which are probably highly localized. Several specific
protein-protein contacts within the complex are described in this
paper. We have studied protein-protein interactions in the complex by
immobilizing individual enzymes and identifying radiolabeled T4
proteins that are retained by columns of these respective affinity
ligands. Elsewhere we have described interactions involving three T4
enzymes found in the complex. In this paper we describe similar
analysis of five more proteins: dihydrofolate reductase,
dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide
reductase, and E. coli nucleoside diphosphokinase,. All eight
proteins analyzed to date retain single-strand DNA-binding protein
(gp32), the product of T4 gene 32. At least one T4 protein, thymidylate
synthase, binds directly to gp32, as shown by affinity chromatographic
analysis of the two purified proteins. Among its several roles, gp32
stabilizes single-strand template DNA ahead of a replicating DNA
polymerase. Our data suggest a model in which dNTP synthetase
complexes, probably more than one per growing DNA chain, are drawn to
replication forks via their affinity for gp32 and hence are localized
so as to produce dNTPs at their sites of utilization, immediately ahead
of growing DNA 3` termini.
For some years our laboratory has investigated interactions
among enzymes of deoxyribonucleoside triphosphate (dNTP) ( Direct support for this
model has been difficult to obtain, because as isolated in purified
form, the dNTP synthetase complex does not contain DNA polymerase or
other replication proteins (Moen et al., 1988). Accordingly,
we have turned to other approaches, including protein affinity
chromatography. In the T4 system, this approach was initially applied
by Formosa et al.(1983) to analysis of interactions involving
gp32, the single strand-specific DNA-binding protein encoded by gene
32. The protein was immobilized, and radiolabeled phage proteins bound
to the chromatographic support were identified by two-dimensional gel
electrophoresis. We have now applied the same approach to immobilized
T4 dCMP hydroxymethylase (gp42), thymidylate synthase (gptd),
and dCMP deaminase (gene cd). Each of these affinity ligands
was found to retain several proteins of the dNTP synthetase complex and
a few proteins of DNA replication and repair/recombination (Wheeler et al., 1992). ( In the present study, we have
immobilized five more proteins: T4 ribonucleotide reductase,
dCTPase-dUTPase, dihydrofolate reductase, deoxyribonucleoside
monophosphokinase, and E. coli nucleoside diphosphokinase,
with results similar to those seen in our earlier analyses.
Unexpectedly, all eight proteins analyzed to date retain gp32 fairly
strongly. Although some of the interactions may be indirect, we found
that immobilized T4 thymidylate synthase tightly binds purified gp32,
indicating a direct interaction between these two proteins. These
observations suggest that dNTP synthetase complexes might be localized
just ahead of growing DNA chains in the replication complex, drawn to
these sites by their affinity for gp32 and functioning there to
maintain local dNTP concentrations sufficient to sustain maximal
replication rates.
During the
course of this work, we found that applying extracts to affinity
columns in the presence of a ``physiological buffer'' led to
binding of higher quantities of protein; the amount of each protein
bound was changed but not the ensemble of bound proteins. (
In our earlier study of
protein interactions with immobilized dCMP hydroxymethylase (gp42), we
considered as significant only proteins that were retained on the
column in 0.2 M NaCl and eluted at 0.6 M NaCl or
higher. Thus, our criterion for significance of binding was more
stringent than that of Formosa et al.(1983). Nevertheless, we
identified 13 proteins that were retained by immobilized gp42 under
these conditions (Wheeler et al., 1992). Subsequently, we
identified six T4 proteins that bind similarly to immobilized
thymidylate synthase (gptd) and eight that bind to dCMP
deaminase (gpcd). Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5show
two-dimensional electrophoretic patterns of T4 proteins strongly
retained by five other enzymes in the T4 dNTP synthetase complex: E. coli nucleoside diphosphokinase (NDP kinase; Fig. 1), aerobic ribonucleotide reductase (Fig. 2),
deoxyribonucleoside monophosphate kinase (dNMP kinase; Fig. 3),
dCTPase-dUTPase (Fig. 4), and dihydrofolate reductase (Fig. 5). Each enzyme behaves similarly to the other three we
have analyzed in binding a half dozen or more T4 proteins specifically
and fairly strongly.
Figure 1:
Proteins in
the 0.6 M NaCl eluate from a column of immobilized E. coli nucleoside diphosphokinase. 8.0 mg of purified NDP kinase was
immobilized, and the elution protocol was carried out by stepwise
addition of NaCl to Tris buffers. Each superscript dot identifies a protein that binds nonspecifically, because it also
binds to immobilized bovine serum albumin or T4
lysozyme.
Figure 2:
Proteins in the 0.5 M NaCl eluate
from a column of immobilized T4 ribonucleotide reductase. 2.0 mg of
purified tetrameric enzyme (products of T4 genes nrdA and nrdB) was immobilized, and elution was carried out by stepwise
addition of NaCl to potassium glutamate buffer, as described under
``Materials and Methods.'' Each superscript dot identifies a nonspecifically bound protein. Upper panel,
standard elution conditions. Lower panel, equilibration of the
column and elution carried out in the presence of 1.0 mM ATP.
Figure 3:
Proteins in the 0.5 M NaCl eluate
from a column of immobilized T4 deoxyribonucleoside monophosphokinase
(gp1). 3.0 mg of purified enzyme was immobilized, and elution was
carried out by stepwise addition of NaCl to potassium glutamate buffer.
Each superscript dot identifies a nonspecifically bound
protein.
Figure 4:
Proteins in the 0.6 M NaCl eluate
from a column of immobilized T4 dCTPase-dUTPase (gp56). 5.0 mg of
purified enzyme was immobilized, and elution was carried out by
stepwise addition of NaCl to Tris buffer. Each superscript dot identifies a nonspecifically bound
protein.
Figure 5:
Proteins in the 0.5 M eluate from
a column of immobilized T4 dihydrofolate reductase (gpfrd).
5.5 mg of purified enzyme was immobilized, and elution was carried out
by stepwise addition of NaCl to potassium glutamate buffer. Each superscript dot identifies a nonspecifically bound
protein.
The results with NDP kinase are particularly
noteworthy, because this enzyme is of bacterial origin, yet a small
amount of NDP kinase is evidently sequestered within the dNTP
synthetase complex by specific protein associations (Reddy and Mathews,
1978; Allen et al., 1983). Even more phage proteins are bound
to this host-cell enzyme if we consider those eluted by 0.2 M NaCl (Fig. 6). These observations are all the more
remarkable when we consider that the immobilized NDP kinase retains
very few E. coli proteins. Fig. 7illustrates this,
showing one-dimensional SDS-polyacrylamide gel electrophoresis analysis
of E. coli and T4 proteins bound to immobilized NDP kinase. Fig. 7C shows one additional observation. Note from Fig. 1that a prominent bound protein is one that we have
identified as the product of gene uvsY, a protein involved in
DNA repair and recombination (Yonesaki et al., 1985). Using
one-dimensional gel analysis, we analyzed T4 proteins in an extract of E. coli infected by a uvsY amber mutant. Several of
the most tightly bound proteins were missing in this pattern (Fig. 7C, far right lane, showing proteins
retained at 0.6 M NaCl but eluted by 2.0 M NaCl). It
seems likely that these proteins do not bind directly to E. coli NDP kinase, but are retained by the column because they associate
with bound gpuvsY.
Figure 6:
Proteins in the 0.2 M NaCl eluate
from a column of immobilized E. coli nucleoside
diphosphokinase. This fraction was collected from the same experiment
described in the legend to Fig. 1. Each superscript dot identifies a nonspecifically bound
protein.
Figure 7:
One-dimensional gel electrophoretic
analysis of proteins bound to immobilized nucleoside diphosphokinase.
Proteins in the 0.2, 0.6, and 2.0 M NaCl eluates were
displayed by one-dimensional SDS-polyacrylamide gel electrophoresis and
autoradiography as described by Wheeler et al.(1992). The far left and far right lanes depict radioactive
molecular weight markers, with the values listed to the right of the figure. The T4 uvsY amber mutant used to prepare
the extract analyzed in part C of the figure was kindly
provided by Dr. Kenneth Kreuzer (Duke
University).
In our analysis of proteins bound to T4
ribonucleotide reductase, we observed the effects of including an
allosteric ligand, ATP, in the eluting buffers. This ligand strongly
affects subunit associations in the heterotetrameric holoenzyme (Hanson
and Mathews, 1994). The lower panel of Fig. 2shows an
electrophoretic pattern from an experiment identical to that of the upper panel, except for the presence of 1 mM ATP in
the column buffer and all of the eluting buffers. This low molecular
weight ligand increased the number and amounts of retained proteins,
suggesting that protein-protein interactions in this complex are
mediated in substantial measure by low molecular weight substrates and
regulatory molecules.
Figure 8:
Retention of purified T4 single-strand
DNA-binding protein by a column of immobilized T4 thymidylate synthase.
0.45 mg of purified gp32 was applied to a 3.0-ml column containing 7.5
mg of thymidylate synthase immobilized on Affi-Gel. Elution was carried
out in potassium glutamate buffer, with stepwise NaCl additions as
indicated. Three fractions were collected in each step. Analysis of
eluates was by one-dimensional SDS-polyacrylamide gel electrophoresis
and Coomassie Blue staining. M
Figure 9:
Immunoprecipitation analysis of polyclonal
antiserum against purified T4 dCTPase-dUTPase (gp56). The method was as
described by Young and Mathews(1992). An extract of
[
Figure 10:
Band shift analysis of protein-protein
interactions. A, analysis with anti-gp1; B, analysis
with anti-dTMP synthase. The protein mixtures were incubated at 1.0
µM each for 30 min at room temperature in the presence of
12.5% polyethylene glycol. A mixture containing 4 µg of total
protein was subjected to nondenaturing electrophoresis in a 10%
polyacrylamide gel. Proteins were then transferred and immunostained
generally as described by Hoffmann et al.(1992). Proteins and
other components added to T4 dNMP kinase (A) or T4 thymidylate
synthase (B) are listed below the respective lanes. dUMP,
where added, was at 1.0 mM. The addition of single-strand DNA
(from M13 phage) was added to the thymidylate synthase reaction
mixtures to facilitate banding of that enzyme. BSA, bovine
serum albumin.
Table 2summarizes the principal results of our
affinity chromatography experiments with eight purified recombinant
proteins that copurify as components of the T4 dNTP synthetase complex.
The table lists proteins identified in two-dimensional electrophoretic
analysis of moderately tightly bound proteins, those that are retained
on the column in 0.2 M NaCl and eluted at 0.6 M NaCl
(or 0.1 and 0.5 M, respectively, when added to a column buffer
already containing 0.1 M potassium glutamate). The
interactions demonstrated with our more limited immunological and band
shift experiments are seen also in results of the affinity
chromatography experiments. All of the protein associations noted here
are consistent with what we have reported in earlier studies (Wheeler et al., 1992). Probably the most remarkable
result of the affinity chromatography experiments is the fact that
gp32, the single strand-specific DNA-binding protein, is retained by
every one of the eight immobilized proteins examined to date. The
associations do not involve the affinity of gp32 for DNA, because the
extracts that we analyze are treated exhaustively with DNase I and
micrococcal nuclease before chromatography. At least one of the
associations is a direct interaction; purified gp32 is retained on a
column of immobilized T4 thymidylate synthase but not by E. coli thymidylate synthase. In this context, one of the unidentified
gp32-binding proteins in the experiments of Formosa et
al.(1983) is almost certainly thymidylate synthase (Wheeler et
al., 1992). In further confirmation of this association, we have
observed interaction of these two proteins in preliminary analytical
ultracentrifugation experiments. ( The physical and
functional relationships between the T4 dNTP synthetase complex and the
DNA replication complex in vivo have long been obscure.
Evidence for existence of the complex was originally sought as a means
to explain how DNA precursors could be delivered to replication sites
at rates sufficient to sustain chain growth rates of 500 s To date, ten enzymes have been reported as
components of the purified dNTP synthetase complex: the eight that we
have immobilized, plus T4 thymidine kinase and E. coli adenylate kinase (Allen et al., 1983; Moen et
al., 1988). From the molecular mass of the purified complex (about
1.5 mDa), it seems that no more than one or two copies of each enzyme
molecule can exist in each complex. However, the turnover numbers for
enzymes in the complex are at least an order of magnitude lower than
the rate of replicative DNA chain growth. Also, the enzyme molecules
themselves exist in considerable molar excess (a few thousand molecules
per cell) over the number of replication forks. By contrast, a
T4-infected cell contains 60 replication forks or 120 growing DNA
chains (Werner, 1968). From the chain growth rates, k The gene 32 protein exists at about 10,000 copies per cell, and it
plays a stoichiometric role in supporting replication; reducing the
number of gp32 molecules per cell reduces the replication rate
proportionately (for reviews, see Karpel(1990) and Kornberg and Baker
(1992)). The number of gp32 molecules associated with each replicating
DNA strand has not been determined, but it must be considerable,
because 10,000 molecules divided by 60 forks divided by two strands per
fork gives about 80 molecules per strand. Because some gp32 molecules
are associated with recombination, DNA repair, and probably other
functions, the number 80 represents an upper limit. However, this
semiquantitative discussion suggests that the associations between gp32
and the dNTP-synthesizing enzymes might provide a route for attracting
several dNTP synthetase complexes to the vicinity of each fork.
Moreover, because gp32 is bound to DNA single strands created by action
of the primosome in advance of the movement of the DNA polymerase
holoenzyme, an association between gp32 and multiple dNTP synthetase
complexes could maintain high dNTP concentrations in the precise region
where they are needed: just in front of a rapidly moving DNA polymerase
holoenzyme. Fig. 11suggests a simplified and speculative form
of this model.
Figure 11:
A speculative model for the association
of T4 dNTP synthetase with DNA replication machinery. For simplicity,
only a leading strand complex is shown. gp43 is DNA
polymerase, and gp45 is the processivity-enhancing protein.
Whether the other polymerase accessory proteins, gp44 and gp62, travel
with the polymerase is not yet established, so they are omitted for
simplicity. The number of complexes per gp32 molecule is
unspecified.
Note from Table 2that several other
replication proteins are bound by enzymes in the dNTP synthetase
complex, including gp61 (primase) and gp45 (processivity-enhancing
protein). Because these proteins were also shown to be bound by
immobilized gp32 (Formosa et al., 1983), the associations with
dNTP-synthesizing enzymes may be indirect; that hypothesis can be
tested experimentally. The associations with gpuvsX and
gpuvsY suggest that the dNTP synthetase complex may be
associated also with the complex that carries out
recombination-dependent replication, because both of those proteins are
involved in that process (Kreuzer and Morrical, 1994). However, the
significance of the associations involving gp If gp32 moves along a replicating
DNA strand by ``treadmilling'' (simultaneous association and
dissociation of individual gp32 molecules), then the model proposed
here is untenable. Association of a 35-kDa protein with a 1.5-mDa
complex would seem to fatally hinder the mobility of a protein that
must be in such continuous motion. However, structural studies on gp32
(Shamoo et al., 1995) indicate that each molecule of the
protein contacts only two or three DNA nucleotide residues. This
finding plus kinetic analysis of gp32 dissociation from DNA in
vitro (Lohman, 1984) are consistent with the idea that the several
gp32 molecules associated with one DNA polymerase holoenzyme slide as a
unit in front of that polymerase. Even so, if gp32 molecules at a
replication fork are associated with large enzyme complexes, then this
implies that a great deal of protein must be moving along with the DNA
polymerase holoenzyme. However, because T4 DNA replication is
associated with the bacterial membrane (Miller, 1972), it is likely
that replication involves movement of DNA chains past a stationary
replicative complex. The data of this paper suggest that that complex
could also include DNA precursor-synthesizing complexes. The
suggestion that gp32 helps to organize dNTP synthetase complexes just
ahead of DNA growing points is speculative. However, it explains
several observations in addition to the affinity chromatographic
analysis described here. More important, the model sets the stage for
future experiments.
Volume 271,
Number 19,
Issue of May 10, 1996 pp. 11156-11162
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)biosynthesis and mechanisms that coordinate dNTP synthesis
with DNA replication (Mathews, 1993a, 1993b). Of particular interest is
the question of how dNTP concentrations are maintained at saturating
levels near replicative DNA polymerases, despite the relentless demand
for precursors created by the extremely high rates of replicative DNA
chain extension (over 500 s
in prokaryotic cells).
In studies of T4 phage-infected Escherichia coli,
Greenberg's laboratory (Chiu et al., 1982) and ours
(Allen et al., 1983; Moen et al., 1988) have
described a multienzyme complex, called dNTP synthetase, which contains
several phage-coded enzymes and at least two enzymes of host cell
origin. In vitro, crude or purified preparations of this
complex display kinetic facilitation of multi-step reaction pathways
leading to dNTPs. In vivo, genetic evidence indicates that DNA
replication draws from precursor pools that are very small in
comparison with the total intracellular dNTP content (Ji and Mathews,
1993). Together, our data support a model in which the T4 dNTP
synthetase complex is localized near replication sites and in which
dNTPs destined for DNA replication are those produced by the complex in
the immediate vicinity of replication forks.
)
Recombinant E. coli Strains
All of the purified
proteins in this study came from expression of recombinant genes
inserted into plasmids and cloned in E. coli. Table 1lists the source of each plasmid. Unless otherwise
indicated, conditions for growth of the bacteria and induction of the
recombinant genes were as described in the references cited.
Purification of Recombinant Proteins
All
recombinant proteins were isolated from induced E. coli cultures of 1-3 liters. All purification schemes yielded
proteins that were at least 95% homogeneous, as estimated by
SDS-polyacrylamide gel electrophoresis.Deoxyribonucleoside Monophosphate Kinase (gp1)
The
fractionation scheme was similar to that of Brush et al. (1990), with some differences. Cells were disrupted by sonic
oscillation rather than French pressure cell treatment. Nucleic acids
were precipitated with protamine sulfate (0.2%) rather than
streptomycin sulfate. In our hands, enzyme precipitated between 30 and
70% saturation with ammonium sulfate rather than 0-45%. Instead
of Cibacron blue affinity chromatography, we used Mono-Q anion exchange
chromatography in a Pharmacia FPLC apparatus with a 0-0.5 M NaCl gradient, in buffer A (50 mM Tris-HCl, pH 7.5, 10
mM 
-mercaptoethanol, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol). Finally, our gel
filtration step used a Superose-12 column and was also carried out in
the FPLC apparatus also in buffer A.dCTPase-dUTPase (gp56)
The plasmid pLAM71* carries
gene 56 downstream from a heat-inducible 
phage promoter.
Induction was carried out by incubation at 42 °C for 30 min. After
collection of the cells by centrifugation, enzyme was isolated by a
scheme based upon that of Warner and Barnes(1966) for the
nonrecombinant gp56. Cells were disrupted by sonic oscillation, and
nucleic acids were precipitated with streptomycin sulfate and then
discarded. These and the subsequent DEAE-cellulose column
chromatography were exactly as described by Warner and Barnes. The next
step, gel exclusion chromatography, was carried out in the FPLC
apparatus with a Superose-12 column. The buffer was 0.2 M potassium phosphate, pH 6.9, containing 1.0 mM -mercaptoethanol and 0.1 M NaCl. The final step,
also carried out in the FPLC apparatus, involved hydrophobic
interaction chromatography on a phenyl-Superose column. The gradient
was from 5 to 0 M NaCl in 0.2 M potassium phosphate
buffer, pH 7.0. Active fractions were pooled and concentrated by
presure dialysis. For further details, see Ungermann(1993).E. coli Nucleoside Diphosphokinase (gpndk)
E.
coli DH5
cells transformed with plasmid pKT8P3 were incubated
overnight in nutrient broth plus 100 µg/ml ampicillin. After
centrifugation, bacteria were resuspended in buffer B (20 mM Tris-HCl, pH 7.4, 10 mM MgCl, 10 mM -mercaptoethanol, 10% glycerol) and disrupted by sonic
oscillation. Fractionation with ammonium sulfate followed with enzyme
precipitating between 45 and 60% of saturation. To the redissolved
protein was added 0.3 volumes of 8% streptomycin sulfate, and the
precipitated nucleic acid was discarded. After dialysis against buffer
C (20 mM Tris-HCl, pH 8.0, 10 mM MgCl, 50
mM KCl, 1 mM -mercaptoethanol, 10% glycerol),
the protein was applied to a 12-ml column of blue Sepharose (Pharmacia
Biotech Inc.) pre-equilibrated in buffer C. NDP kinase was specifically
eluted by 2 mM thymidine diphosphate in distilled water. After
pressure dialysis, the concentrated material was loaded onto a Mono-Q
FPLC column, and elution was carried out in a 0-0.5 M KCl gradient in buffer C. For complete details, see Ray(1992).Dihydrofolate Reductase (gpfrd)
This procedure was
based upon one originated in our laboratory (Purohit and Mathews,
1984). Bacteria were disrupted by sonic oscillation, and nucleic acids
were precipitated from the extract with 0.3 volumes of 7% streptomycin
sulfate. Enzyme was precipitated by the addition of ammonium sulfate to
45% of saturation. Hydrophobic interaction chromatography followed with
a gradient of 1.7-0 M ammonium sulfate in 50 mM Tris-HCl buffer, pH 7.0, containing 10 mM -mercaptoethanol. Active fractions were pooled, dialyzed, and
applied to a Mono-Q column in the FPLC apparatus and then eluted with a
0-0.4 M NaCl gradient in 50 mM Tris-HCl, pH
7.0.Ribonucleotide Reductase (gpnrdA and
nrdB)
Induction of bacteria containing the pnrdAB
plasmid was carried out by growing a 1-liter culture in LB broth plus
100 µg/ml ampicillin to an A
of about 0.7
and adding to that culture 400 µM isopropyl-thiogalactoside and 10 µM ferrous ammonium
sulfate. After 4-6 h of induction at 30 °C, cells were
collected by centrifugation and suspended in 50 mM Tris-HCl,
pH 9.0, 20 mM NaCl, 5 mM MgCl, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM ferrous ammonium sulfate (buffer
D). Cells were disrupted by sonic oscillation, and nucleic acids were
precipitated from the clear supernatant by adjustment to 1.0%
streptomycin sulfate. Ammonium sulfate was added to 40% of saturation,
and the precipitated protein was dissolved in buffer D and desalted
into the same buffer. Chromatography was carried out in a Mono-Q
anion-exchange column in the FPLC apparatus, using a gradient of
40-200 mM NaCl in buffer D. This column separated the
R1R2 holoenzyme from its constituent unassociated R1 and R2 subunits. A
typical 1.0-liter culture yielded about 1 mg of free R1 subunit, 2.5 mg
of free R2 subunit, and 4.5 mg of R1R2 holoenzyme. For complete
details, see Hanson(1994).Thymidylate Synthase (gptd)
Recombinant T4
thymidylate synthase was prepared as described elsewhere.Single-strand DNA-binding Protein
(gp32)
This procedure was a modification (
)of a
method described by Shamoo et al.(1986). Gene 32 expression
was induced in E. coli AR120 carrying plasmid pYS6 by
overnight growth at 37 °C in the presence of 0.12 mg/ml nalidixic
acid. After centrifugation, cells were resuspended in 3 volumes of
buffer E (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1
mM EDTA, 0.1 mM -mercaptoethanol, 10% glycerol)
plus 10 mM MgCl and 2 mM CaCl. After sonic disruption of the cells, DNase I was
added to 20 µg/ml, and the lysate was incubated on ice for 30 min.
After centrifugation, 5 mM EDTA was added to the supernatant
(and to all buffers used subsequently), which was then loaded onto a
DEAE-cellulose column pre-equilibrated with buffer E and then eluted
with an NaCl gradient of 0.05-0.5 M in buffer E.
Fractions containing gp32 were pooled, dialyzed against buffer E, and
applied to a single-strand DNA-cellulose column pre-equilibrated in
buffer E. The column was washed with 0.5 M NaCl in buffer E,
and gp32 was displaced by a step elution with 2.0 M NaCl in
buffer E.Affinity Chromatography
Methods for immobilization
of protein, radiolabeling of T4 proteins, elution of proteins from
affinity columns, and identification of retained proteins after
two-dimensional gel electrophoresis were generally as described
previously (Wheeler et al., 1992). Briefly, T4 proteins were
labeled by incorporation of [
S]methionine from 3
to 8 min after infection of E. coli B at 37 °C. An extract
was applied in column buffer to a column prepared by immobilizing
2-5 mg of purified recombinant enzyme on Affi-Gel-10 (Bio-Rad).
Column buffer is 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1
mM -mercaptoethanol, 5 mM MgCl,
0.025 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride,
and 10% glycerol. After thorough washing to remove unbound,
``flow-through'' proteins, bound proteins were eluted in
three steps: first, by 0.2 M NaCl in column buffer, next by
0.6 M NaCl, and finally by 2.0 M NaCl.
)The physiological buffer contains potassium and glutamate,
reflecting the principal intracellular small ions in E. coli (Richey et al., 1987). That buffer contains 0.1 M potassium glutamate, pH 8.0, 10% glycerol, 0.5 mM magnesium acetate, 1.0 mM -mercaptoethanol, and 0. 2
mM phenylmethylsulfonyl fluoride. Elution involved addition of
NaCl to this modified column buffer in steps of 0.1, 0.5, and 2.0 M. We identify the conditions used for chromatography in each
of the respective figure legends.
Two-dimensional Gel Electrophoretic Analysis of Bound
Proteins
Formosa, Burke, and Alberts(1983) originally used
protein affinity chromatography to identify protein-protein
interactions in T4 phage infection. That study used gene 32 protein as
the affinity ligand, and each interacting protein was identified by two
criteria: 1) retention on the column at 0.05 M NaCl and
elution at 0.2 M NaCl and 2) failure to be similarly retained
by a column of immobilized bovine serum albumin. We have also used
bovine serum albumin as a negative control; in addition, we have found
that the same set of nonspecific proteins binds to a column of
immobilized T4 lysozyme (data not shown).
A Direct Interaction between gp32 and T4 Thymidylate
Synthase
Table 2lists all of the T4 proteins that we have
identified in the 0.6 M NaCl eluates as being bound
specifically to each of the eight immobilized enzymes that we have
examined. Remarkably, one protein retained by all eight affinity
ligands is gp32, the single-strand DNA-binding protein. This finding is
particularly noteworthy, because gp32, a protein of moderate size, has
already been shown to interact with numerous proteins of DNA
replication, recombination, and repair (Formosa et al., 1983;
Krassa et al., 1991; Hurley et al., 1993). It seems
unlikely that gp32 binds directly to each of the eight enzymes we have
examined. Rather, some of these proteins probably associate indirectly
by binding to a protein that binds directly to gp32. In fact, our
earlier study (Wheeler et al., 1992) presented evidence
supporting a direct association between gp32 and T4 thymidylate
synthase. We tested that idea by asking whether purified gp32 is
retained on a column of immobilized T4 thymidylate synthase. As shown
in Fig. 8, some of the gp32 was found in the flow-through
fractions, suggesting that the binding capacity of the immobilized dTMP
synthase column had been exceeded. However, of that protein that did
bind, most was retained at 0.2 M NaCl and eluted at 0.6 M, indicating a strong interaction between these proteins in
the absence of other proteins. When the same experiment was repeated
with immobilized E. coli dTMP synthase instead of the T4
enzyme, no retention of gp32 was seen, indicating the specific nature
of the interaction between the two T4 proteins (data not shown).
, molecular weight
markers; gp32, purified protein applied to the column; FT, flow-through fraction.
Other Protein-Protein Associations in the dNTP Synthetase
Complex
The original aim of this study was not, as implied by
the title of this paper, to characterize interactions between the dNTP
synthetase complex and gp32. Rather, our goal was (and remains) to
identify as many protein-protein interactions as possible, using a
range of experimental approaches. Several other interesting
associations have been discovered recently, and they are described in
this section.An Interaction between dCTPase-dUTPase and dNMP
Kinase
In earlier studies we have used anti-idiotypic antibodies
as probes to identify interacting proteins. For example, antibodies to
antibodies against T4 dCMP hydroxymethylase reacted with T4 dTMP
synthase, supporting an interaction between these two proteins (Young
and Mathews, 1992). In the present study, we applied this approach to
the dCTPasedUTPase encoded by T4 gene 56. Unexpectedly, we found that
polyclonal antiserum against purified gp56 reacted with two T4
proteins, as shown by immunoprecipitation. As shown in Fig. 9,
one protein had an M of 20,000, close to that of
gp56. The other had an M of about 26,000. Because
this latter molecular weight corresponds closely to that of gp1, the
trifunctional dNMP kinase, we hypothesized that the injected gp56 was
so strongly antigenic that an anti-idiotypic response had occurred in
the rabbit undergoing the primary immunization and that this had
generated antibodies directed against gp1. Alternatively, an
association between gp56 and gp1 is so strong that anti-gp56 antibodies
precipitated gp1 bound to gp56. The identities of the two proteins were
tested by adding each respective protein in purified form to the
immunoprecipitation mixtures to dilute the specific radioactivity of
each protein. As shown in Fig. 9, the addition of purified gp56
caused the 20-kDa band to disappear, and the addition of purified gp1
caused the 26-kDa band to disappear. Later, anti-idiotypic antibodies
to gp56 were prepared by purifying anti-gp56 antibodies on a gp56
column and injecting those antibodies into naive rabbits. Antiserum
from one of these animals also precipitated gp1, as shown by a dilution
experiment like that of Fig. 9(data not shown). Thus, by these
criteria, gp1 and gp56 interact directly.
S]methionine-labeled T4 proteins (3-8 min
after T4 infection) was incubated with serum, and the
immunoprecipitates were analyzed by one-dimensional SDS-polyacrylamide
gel electrophoresis and autoradiography. Lane 1, preimmune
serum; lane 2, antiserum; lane 3, gp56 dilution
experiment: antiserum plus 20 µg of purified gp56 added to the
incubation mixture; lane 4, gp1 dilution experiment: antiserum
plus 20 µg of purified gp1 added to incubation mixture; lane
5, identical to lane 2.
Protein Associations as Revealed by Band Shift
Assays
Hoffmann et al.(1992) have described the use of
nondenaturing gel electrophoresis to identify pairwise interactions
between purified proteins. In the experiment of Fig. 10A, we used this approach to confirm the
interaction between dNMP kinase and dCTPase-dUTPase. After native
polyacrylamide electrophoresis, proteins on the gel were transferred to
nitrocellulose and probed with antibodies to purified dNMP kinase.
Although the band shift seen in the presence of dCTPase-dUTPase was not
large, it was significant compared with the migration of a mixture of
bovine serum albumin and dNMP kinase (compare third and fourth lanes). The experiment of Fig. 10B presents comparable data supporting an interaction between T4
thymidylate synthase and dihydrofolate reductase. The band shift became
even more pronounced when dCTPase-dUTPase was added to the mixture,
suggesting that the three proteins interact. However, the thymidylate
synthase substrate, dUMP, apparently disrupted these interactions
(compare third and fifth lanes of Fig. 10B).
) or more. However, isolation of the complex provided little
evidence for its association with replication proteins, although DNA
polymerase was observed to cosediment with dNTP-synthesizing enzymes in
gradient analysis of T4-infected cell extracts (Chiu et al.,
1982). On the other hand, genetic evidence indicates that dNTPs used
for replication are drawn from pools that are much smaller than those
determined by biochemical analysis (Ji and Mathews, 1993), as expected
if growing DNA chains draw precursors from dNTPs synthesized in the
immediate vicinity.
values for the enzymes involved, and the base composition of T4
DNA and assuming that k values in vivo are comparable with those for the purified enzymes, we can
estimate that 10-20 molecules of each dNTP-synthesizing enzyme
are needed to serve each DNA chain. Thus, if the dNTP synthetase
complex facilitates the transfer of dNTPs to replication sites, several
complexes must serve each replication site. How might this occur?
gt is difficult to
discern at this stage, because the reaction catalyzed by the gene
product, glucosylation of DNA hydroxymethyl-cytosine residues, occurs
away from the replication fork.
)))))
We thank those colleagues identified in Table 1who provided clones and purified proteins. We are grateful
to Melissa Clason for purification of dNMP kinase. We thank Dr. Karl
Drlica (Public Health Research Institute) for keen insights and useful
suggestions regarding this manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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