(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) ()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.
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. ()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.
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, molecular weight
markers; gp32, purified protein applied to the column; FT, flow-through fraction.
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
[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.
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 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.
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 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?
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 gpgt 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.
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.