From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, February 15, 2001
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ABSTRACT |
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The lagging strand of the replication fork is
initially copied as short Okazaki fragments produced by the coupled
activities of two template-dependent enzymes, a primase
that synthesizes RNA primers and a DNA polymerase that elongates them.
Gene 4 of bacteriophage T7 encodes a bifunctional
primase-helicase that assembles into a ring-shaped hexamer with both
DNA unwinding and primer synthesis activities. The primase is also
required for the utilization of RNA primers by T7 DNA polymerase. It is
not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the
minimal requirements for RNA primer utilization by T7 DNA polymerase,
we created an altered gene 4 protein that does not form
functional hexamers and consequently lacks detectable DNA unwinding
activity. Remarkably, this monomeric primase readily primes DNA
synthesis by T7 DNA polymerase on single-stranded templates. The
monomeric gene 4 protein forms a specific and stable complex with T7
DNA polymerase and thereby delivers the RNA primer to the polymerase
for the onset of DNA synthesis. These results show that a single
subunit of the primase-helicase hexamer contains all of the residues
required for primer synthesis and for utilization of primers by T7 DNA polymerase.
DNA replication is mediated by a complex of proteins that
assembles at the replication fork and directs the coordinated synthesis of two DNA strands. Four proteins account for the major reactions occurring at the replication fork of bacteriophage T7 as follows: the gene 5 DNA polymerase and its processivity factor,
Escherichia coli thioredoxin, the gene 2.5 single-stranded
DNA-binding protein, and the gene 4 primase-helicase (1). The
activities of these replication proteins are coordinated by their
physical interactions during replication, which serve to couple the
synthesis of the leading strand with that of the lagging strand (2).
The primase-helicase is a fixture of the T7 replisome that directly
contacts both the DNA polymerase and the single-stranded DNA-binding
protein (2-6). A C-terminal acidic segment of the primase-helicase is
required for its interaction with T7 DNA polymerase (4). This stable protein-protein interaction could correspond to an interaction between
the helicase bound to the lagging strand of the replication fork and
the polymerase on the leading strand.
As the replication fork moves along a DNA duplex, DNA primase
periodically deposits short RNA primers at specific priming sequences
on the lagging strand, triggering the synthesis of Okazaki fragments
that are subsequently processed to form a continuous DNA strand (7, 8).
In most DNA replication systems, a separate primase protein transiently
interacts with the DNA helicase to initiate primer synthesis on the
lagging strand. In E. coli, the strength of this interaction
affects the frequency of priming and thereby sets the average length of
Okazaki fragments (9). The primase and helicase activities of
bacteriophage T7 are fused in a single polypeptide that assembles into
a ring-shaped hexamer (10-12). The bifunctional primase-helicase
unwinds DNA ahead of the replication fork, and it primes the
discontinuous synthesis of the lagging strand of the replication fork.
The short tetranucleotides synthesized by the primase domain of the
primase-helicase are not extended by T7 DNA polymerase alone (13-15);
they are elongated by the polymerase only if the primase-helicase is
also present during primer extension. It is not known how many subunits
of the hexameric primase-helicase directly participate in the priming of DNA synthesis, nor is it known how the primase-helicase stimulates primer utilization by T7 DNA polymerase. The primase-helicase protein
consists of an N-terminal primase domain and C-terminal helicase domain
(12, 16, 17) that will separately catalyze tetraribonucleotide
synthesis and DNA unwinding, respectively (16, 18, 19). However, the
primase domain alone does not support the extension of primers by T7
DNA polymerase (18). The dual requirement for the primase-helicase and
the T7 DNA polymerase during RNA-primed synthesis of DNA suggests that
these proteins associate in a complex that initiates the elongation of
RNA primers synthesized by the primase (20).
The ring-shaped T7 primase-helicase catalyzes DNA unwinding by
encircling one strand of DNA (12, 21) and moving in a 5' to 3'
direction along one DNA strand while displacing the complementary strand (22-26). The vectorial movement of the protein on DNA is coupled to the hydrolysis of 2'-deoxythymidine triphosphate (dTTP) (25,
27, 28), and thus, the helicase is a type of molecular motor. Crystal
structures of the helicase domain of the primase-helicase (29, 30)
revealed that the nucleotide-binding sites are located at the
interfaces between subunits of the hexamer, where changes in the
relative orientations of the subunits could influence the catalytic
activities of the six potential active sites within the helicase. Three
different relative orientations of adjacent subunits are observed in
the hexameric helicase that was crystallized, and these different
orientations affect the nucleotide binding properties of individual
subunits (30). This conformational flexibility, together with previous
biochemical and genetic data revealing the identities of functionally
important residues and the cooperative behaviors of nucleotide binding
and hydrolysis by the hexameric helicase, are the basis for several
proposed mechanisms of DNA unwinding (30, 31). In these models,
nucleotide hydrolysis is coupled to changes in protein conformation and
DNA binding affinity that allow the protein to step along DNA, not unlike other motor proteins that travel along protein filaments in
response to nucleotide hydrolysis (32, 33). Some aspects of the
proposed mechanisms of DNA unwinding resemble those of the bind-change
mechanism of rotary catalysis proposed for the mitochondrial
F1-ATPase (34, 35).
There are currently few high resolution structures of primases, and
none with substrates bound (36-38). The DNA binding and catalytic
properties of the T7 primase are well characterized, making it an
attractive candidate for structural analysis. Like the intact
primase-helicase, a primase fragment (residues 1-271) of the T7
primase-helicase catalyzes the template-dependent synthesis of RNA oligomers at specific priming sites as follows:
5'-(G/T)(G/T)GTC-3' (18, 39, 40), making predominantly pppAC,
pppACC(C/A), and pppACAC. The conserved 3'-C of the priming sites is
required for primer synthesis, but it is not copied into the RNA
products. The tetraribonucleotides synthesized by an isolated primase
fragment are bona fide primers that can be extended by T7
DNA polymerase, provided the intact primase-helicase protein is added
during primer extension (18). The primase domain fragment of the T7
primase-helicase is monomeric even at very high protein concentrations,
and its failure to support primer utilization by T7 DNA polymerase
suggested that several subunits of the hexameric primase-helicase might cooperate to deliver tetranucleotide primers to the polymerase. Such
cooperation could occur either by recruiting the polymerase through
protein-protein interactions or by preventing dissociation of the RNA
primer from the DNA template by sequestering the primer in a stable
protein-DNA complex. A long lived complex of the primase-helicase and
M13 single-stranded DNA forms in the presence of ribonucleotide substrates for primer synthesis (13). This primase-helicase-DNA complex
most likely contains the ribonucleotide product of the primase annealed
to DNA and ready for elongation by T7 DNA polymerase (14).
We wished to determine if a single subunit of the primase-helicase
could provide all of the necessary DNA binding and protein contacts for
priming synthesis of DNA by T7 DNA polymerase. In order to define the
minimal requirements for primer utilization by T7 DNA polymerase, we
have genetically engineered an altered primase-helicase that does not
assemble into functional hexamers and therefore lacks DNA unwinding
activity. We show that the monomeric primase physically associates with
T7 DNA polymerase in a protein-DNA complex that initiates RNA-primed
synthesis of DNA.
Materials--
The oligonucleotides used in these studies were
synthesized on an Applied Biosystems model 394 DNA synthesizer and
purified by high performance liquid chromatography using an HQ20 anion exchange column (Applied Biosystems) or by gel electrophoresis using
20% acrylamide, 7 M urea gels. The T7 primase-helicase and T7 DNA polymerase (a 1:1 complex of T7 gene 5 protein and E. coli thioredoxin) were purified by published procedures (41, 42). A variant of T7 DNA polymerase with two amino acid substitutions in the
active site of the 3'-5'-exonuclease (Asp-5 Mutagenesis of the T7 Primase-Helicase--
The mutant T7 gene 4 protein, gp4 Preparation of gp4 Nondenaturing Gel Electrophoresis--
Native PAGE was performed
in a 15% acrylamide Tris-glycine gel (Bio-Rad) with a Mini-PROTEAN II
electrophoresis system (Bio-Rad). The electrophoresis buffer was 25 mM Tris-HCl, 190 mM glycine, 5 mM
MgCl2, with 1 mM ATP added to stabilize the
hexameric form of the primase-helicase (10, 19). Protein samples (10 µM monomer) in 20 mM Tris-HCl (pH 7.5), 10 mM DTT, 5 mM MgCl2, 5 mM dTTP Hydrolysis--
The dTTPase activity of the helicase domain
of the primase-helicase is a sensitive indicator of hexamer formation
(19) because the active site of the helicase is located at the
interface between subunits of the hexamer (29). Hydrolysis of dTTP was
monitored using thin layer chromatography as described previously (11). The 10-µl reaction mixture contained 30 mM Tris-HCl (pH
7.5), 10 mM MgCl2, 10 mM DTT, 5 mM dTTP (including 5 µCi of [3H] dTTP), and
the indicated concentrations of the wild-type primase-helicase or of
gp4 Helicase Activity Assays--
The helicase activity of the T7
primase-helicase was measured with two different assays, either by
monitoring the enzymatic separation of two DNA strands (27) or by its
ability to support DNA replication in a rolling circle DNA replication
reaction (2). DNA strand separation activity was monitored as the
dissociation of a 5'-32P-labeled 37-mer oligonucleotide
(5'-TCACGACGTTGTAAAACGACGGCCAGTTTTTTTTTTT-3') annealed to M13 ssDNA
(11). The oligo(dT) sequence at the 3' end of the oligonucleotide is
not complementary to M13 ssDNA, which allows the
primase-helicase to load onto the M13 DNA at the junction between
single-stranded and double-stranded regions. The helicase catalyzed
strand separation in a 5'-3' direction along the circular M13 DNA
template. The 10-µl reaction mixture contained 40 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 50 µg/ml BSA, 5 mM dTTP, 20 ng/µl
DNA substrate (described above), and the indicated concentrations of
the primase-helicase. The mixture was incubated for 15 min at 23 °C,
and then the reaction was stopped by adding 2 µl of 500 mM EDTA. The samples were loaded on the 15% nondenaturing
polyacrylamide gel with TBE buffer (90 mM Tris-HCl, 90 mM borate, 2 mM EDTA (pH 8.0)) and
electrophoresed at 300 V for 2 h at an ambient temperature of
4 °C. The amounts of radiolabeled oligonucleotide remaining annealed
to the substrate or in the dissociated product strand were visualized
by autoradiography.
We also examined the ability of the primase-helicase to stimulate DNA
synthesis by T7 DNA polymerase on a duplex DNA template in a reaction
that requires DNA strand separation by the helicase (2). The DNA
template for this assay was a circular 70-nucleotide DNA template
annealed to an oligonucleotide with a 3'-hydroxyl annealed to the
template and an unpaired 5' end that facilitates the loading of the
primase-helicase onto DNA (Fig. 4, inset). The sequence of
this substrate was designed to identify and quantitate specifically the
leading strand synthesis that is linked to the DNA unwinding by
measuring the incorporation of [ Oligoribonucleotide Synthesis--
Oligoribonucleotides
synthesized by the primase-helicase on a synthetic oligonucleotide
(5'-CAGTGACGGGTCGTTTATCGTCGGC-3'; template 3 shown in Table
IV) were analyzed by gel electrophoresis. The reaction mixture (10 µl) contained 40 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, 10 mM DTT, 100 µg/ml BSA, 50 mM potassium glutamate, 0.1 mM 25-mer template,
1 mM each of [ RNA-primed DNA Synthesis--
The synthesis of
tetraribonucleotides by the primase-helicase and their elongation by T7
DNA polymerase were measured as described previously (4). The reaction
mixture (10 µl) contained 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 100 mg/ml BSA,
50 mM potassium glutamate, 25 ng/µl M13 ssDNA, 1 mM each of ATP and CTP, 0.3 mM each of dATP,
dCTP, and dGTP, 0.3 mM [3H]dTTP, 100 nM of exo Formation of a Priming Complex--
We examined the stability of
a protein-DNA complex consisting of gp4
To determine the effect of primer length on the stability of the
priming complex, the compositions of 2'-deoxynucleotide(s) and
2',3'-dideoxynucleotide were adjusted in the primer synthesis reaction
to create RNA-DNA products of the desired lengths. The appropriate dNTP
(10 mM) complementary to the template nucleotide adjacent
to the primer 3' end was then added to each protein-DNA complex prior
to challenge with exonuclease III. For example, the complex with a
5-mer (pppACCCddG) primer strand was prepared with ATP, CTP, and ddGTP
for the primer synthesis reaction, followed by the addition of dTTP for
the assembly of the stable priming complex. DNA templates of different
lengths (Table IV) were examined using similar methods.
To determine the contribution of the 5'-triphosphate moiety of the
primer to the stability of the priming complex, primer synthesis was
initiated with a preformed diribonucleotide triphosphate (pppAC), or an
unphosphorylated diribonucleotide (AC), in a reaction that included
[ Isolation of a Protein Complex That Mediates Primer
Utilization--
The protein-DNA complex containing the
primase-helicase and T7 DNA polymerase was isolated by affinity capture
on an immobilized DNA template (template 3 in Table IV) containing a
3'-biotin group bound to avidin-agarose beads. To form the immobilized
complex, a 6-mer primer strand was enzymatically synthesized using the reaction conditions described above, and then the reaction was diluted
10-fold into a binding solution (10 µl) consisting of 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
10 mM dCTP, 10 mM DTT, 50 mM
potassium glutamate, 0.2% Tween 20, 3.5 µl of NeutrAvidin-agarose resin (Pierce), and 10 µM each of gp4 Construction of a Monomeric Primase-Helicase--
In order to
study the minimal requirements for primer utilization by T7 DNA
polymerase, we constructed a monomeric variant of the T7
primase-helicase. Crystal structures of the helicase domain of the
primase-helicase (29, 30) have revealed an interlocking arrangement of
neighboring subunits within the hexamer. The N-terminal
Our strategy to prevent oligomerization of the primase-helicase was to
eliminate two helices located at the subunit interface (helices D2 and
D3; residues 368-382), and to replace this segment with a short polar
sequence (NH2-SASASG-COOH; Fig. 1a) that would be unlikely to stabilize the hexameric packing seen in the crystal structure (29, 30). The resulting 61-kDa protein, which we have named
gp4
The oligomeric state of gp4 gp4
gp4 gp4
The tetraribonucleotides synthesized by the T7 primase-helicase can be
extended by T7 DNA polymerase only in the presence of the
primase-helicase (26, 28). Although a fragment spanning the N-terminal
half of the primase-helicase correctly synthesizes tetraribonucleotides, the primase fragment does not support their utilization by T7 DNA polymerase (18). The primase fragment and
gp4 A Priming Complex of gp4
The association of the hexameric T7 primase with T7 DNA polymerase
during extension of a tetraribonucleotide primer was previously demonstrated using an exonuclease challenge assay (20). We investigated the minimal requirements for primer utilization by T7 DNA polymerase, using the monomeric gp4
A different type of interaction between gp4 and T7 DNA polymerase has
been described (4) that requires an acidic segment at the C terminus of
the helicase domain. The analogous C-terminal region of gp4
Several factors strongly influence the stability of the T7 priming
complex. A high concentration of the incoming dCTP substrate is
required to keep the polymerase bound to the primed DNA template (Fig.
7) (45). The priming complex becomes more
stable as the concentration of dCTP is increased, reaching maximal
stability at 5-10 mM dCTP (Table I). Although dCTP is the
correct incoming nucleotide specified by the template, the binding of
several mismatched dNTP substrates in the polymerase active site
measurably stabilizes the complex (Fig.
8). For these experiments, the polymerase
incorporated [
The length of the extended primer strand has a pronounced effect on the
lifetime of the complex. The long lived complex described above
contains an elongated primer consisting of 6 nucleotides (5'-pppACCCdGddT-3') that is annealed to a 25-mer template (template 3 in Table IV). Similar priming complexes were prepared by halting primer
elongation after the incorporation of 1-4 deoxynucleotides by T7 DNA
polymerase, and the lifetimes of these complexes were determined by
challenge with exonuclease III. The complex with a single
dideoxynucleotide appended to the tetraribonucleotide (the 5-mer
complex in Fig. 9) is less stable than
the 6-mer complex described above, and it has a half-life of about 30 min (Table III). The 6-mer complex is
most stable, with a half-life of more than 60 min (Figs. 6b
and 9; Table III). Further elongation of the primer strand to create
the 7- and 8-mer complexes incrementally decreases the stability of the
priming complex (Fig. 9 and Table III). The destabilization of the
priming complex with increasing length of the DNA product might
indicate that interactions between the primase and polymerase are
weakened as the polymerase moves away from the priming site during DNA
synthesis. The elongating primer strand eventually becomes effectively
long enough that the primase is no longer needed for DNA synthesis.
The priming complexes described above were bound to a 25-mer DNA
template (template 3 in Table IV). We
also examined DNA templates 19-31 nucleotides in length with the same
priming sequence in complexes containing the 6-mer elongation product
(pppACCCdGddT) and the bound nucleotide dCTP. All of these templates
supported the synthesis of tetraribonucleotides by gp4 The 5'-Triphosphate of the Primer Stabilizes the Priming
Complex--
The ribonucleotides synthesized by the T7 primase contain
a 5'-triphosphate moiety from the ATP that initiates synthesis. The
primase can also incorporate ATP analogs with chemically modified phosphate linkages at the 5' end of ribonucleotide products (39). This
relaxed specificity suggests that the primase does not interact strongly with the 5'-triphosphate moiety during RNA synthesis. The
unphosphorylated dinucleotide AC is also efficiently extended by the
primase to form a tetraribonucleotide (ACCC) that is utilized by T7 DNA
polymerase (14). Thus, the role of the 5'-triphosphate of the naturally
occurring products of T7 primase is enigmatic. We examined whether or
not the 5'-triphosphate contributes to the physical stability of
priming complexes formed between gp4 Purification of the Priming Complex Using an Affinity-tagged
Template--
The exonuclease III protection assay described above
provided indirect evidence that the T7 primase and the polymerase were stably associated in the priming complex. In order to observe the
complex directly, we purified the complex by a biotin-avidin affinity
capture procedure. The priming complex was assembled on a
3'-biotinylated template (template 3 in Table IV) coupled to
avidin-agarose beads. The beads were washed to remove proteins bound
nonspecifically, and the remaining proteins complexed to the template
were eluted with SDS and visualized by SDS-PAGE (Fig. 11). In the complete priming reaction
(lane 2) both gp4 As the replication fork advances, RNA primers are periodically
synthesized by a primase on the lagging strand and extended by a DNA
polymerase to create Okazaki fragments several hundred to several
thousand nucleotides in length (8). The priming sites used by
prokaryotic replication systems are short sequences that are expected
to occur by chance every several hundred nucleotides. However, not
every potential priming site is used, and it has been suggested that a
timing mechanism regulates the frequency of primer synthesis and hence
the length of Okazaki fragments produced during replication. In
E. coli, the DnaG primase transiently associates with the
replication machinery to prime Okazaki fragment synthesis, and then the
primase dissociates from the replication complex. The strength of this
interaction between the primase and helicase might serve as a timing
mechanism that controls the frequency of priming (9). The consolidation
of primase and helicase functions in the bifunctional primase-helicase
of bacteriophage T7 raises questions about how many subunits of the
hexamer participate in primer synthesis and utilization by T7 DNA
polymerase, and how the frequency of these events is controlled during replication.
We have engineered an altered T7 primase-helicase (gp4 gp4 The finding that the stability of the priming complex decreases as the
primer strand is elongated (Fig. 9 and Table III) supports the notion
of a direct interaction between the primase and the polymerase within
the priming complex. Interactions with the 5'-triphosphate of the
primer, which contribute substantially to the stability of the complex
(Fig. 10 and Table V), could fix the location of the primase on the DNA
template. T7 DNA polymerase engages the other (3') end of the growing
primer strand within the priming complex, as shown by the absolute
requirement for a dNTP substrate to stabilize the priming complex. The
length of the primer strand, and hence the distance between the primase
and polymerase proteins bound to either end of the short primer strand,
is a critical determinant of the stability of the priming complex (Fig.
10), implying a direct interaction between these proteins. Longer DNA primers are utilized by T7 DNA polymerase without assistance from the
primase. In the crystal structure of the polymerizing complex (45), the
DNA binding groove of the polymerase makes numerous contacts with DNA
duplex exiting the polymerase. For the utilization of
tetraribonucleotide primers, the primase protein might serve as a
surrogate for these DNA contacts until the primer becomes sufficiently
long to fill the DNA binding groove of the polymerase. This model
suggests that the primase protein partially occupies the DNA binding
groove of the polymerase, where it secures the RNA primer in the
polymerase active site. A second role of the primase might be to
prevent the dissociation of its short RNA product from the template by
remaining bound to the nascent primer prior to its engagement by T7 DNA
polymerase (3).
The formation of a stable priming complex requires a high concentration
of an incoming nucleotide that can pair with the next base of the
single-stranded DNA template in the active site of the polymerase (Fig.
7 and Table I). Although the nucleotide that can correctly pair with
the template generates the most stable priming complex, other
mismatched nucleotides can also stabilize the complex to varying
extents (Fig. 8 and Table II). In particular, dTTP is able to pair with
a template guanosine to produce a fairly long lived complex, although
this complex cannot be isolated by the affinity capture procedure shown
in Fig. 11. The addition of dGTP in combination with a guanine template
base does not generate a stable complex (Fig. 8). In contrast, addition
of dATP provides some protection in the exonuclease challenge assay
(Fig. 8). The oversized guanine-guanine pair is apparently excluded
from the polymerase active site, whereas an adenine-guanine pair might form a more favorable syn/anti base pairing geometry
(54).
We have identified the minimal protein requirements for primer
utilization by T7 DNA polymerase. A monomeric gene 4 protein lacking
helicase activity is able to prime DNA synthesis by the polymerase. If
DNA synthesis is halted during the first few cycles of nucleotide
incorporation by the polymerase, the monomeric primase remains
associated in complex with the polymerase on the primed DNA substrate.
These interactions allow short RNA primers, which otherwise are not
recognized by the polymerase, to be utilized and extended. The stalled
primer elongation complex described here is an attractive candidate for
structural analyses of interactions mediating the initial steps of DNA
synthesis on the lagging strand of the T7 replication fork.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala and
Glu-7
Ala; constructed by Stanley Tabor) was used for the
assembly of stable protein-DNA complexes. This altered DNA polymerase
has a wild-type level of DNA polymerase activity, but the exonuclease activity of the altered polymerase is reduced by a factor of
106 (43). We therefore refer to this modified polymerase as
exo
T7 DNA polymerase. The primase fragment (residues
1-271) of the T7 primase-helicase was purified as described previously
(18). E. coli DH5
was obtained from Life Technologies,
Inc., and E. coli BL21(DE3) was obtained from Novagen. M13
single-stranded DNA (ssDNA)1
and restriction enzymes were obtained from New England Biolabs. dNTPs
were purchased from Promega Corp. ATP, CTP, and all radiolabeled materials were from Amersham Pharmacia Biotech. The diribonucleotide ApC was obtained from Sigmao. The ribonucleoside triphosphate pppApC
was prepared as described under "Exonuclease III Protection Assay" below.
D2D3, has the sequence SASASG substituted for residues
368-382 of the primase-helicase, and consequently, it is nine residues
shorter than the wild-type protein (Fig. 1). This amino acid
substitution eliminates two
-helices (helices D2 and D3) located at
the subunit interface of the helicase domain of the primase-helicase
(29, 30) and thus should prevent the formation of the hexamer. An
expression plasmid for gp4
D2D3 was constructed by cloning a
BsaI-AflII fragment containing the desired
modifications from the plasmid m4D1 (encoding a C-terminal
fragment of the gene 4 protein, provided by Leo Guo, Harvard Medical
School) into an expression plasmid for the full-length primase-helicase, pETgp4A'-A (obtained from David Frick, Harvard Medical School). The resulting plasmid, pGP4
D2D3, is based on the
pET24a vector (Novagen Inc.), and it places the gene encoding the
modified primase-helicase under control of a T7 promoter with a binding
site for the lac repressor, which in turn suppresses background
expression of protein prior to induction. Codon 64 of the gp4
D2D3
coding sequence has been changed from methionine to a glycine codon in
order to prevent internal initiation of translation at codon 64 (44).
D2D3--
A 5-ml overnight culture of
E. coli BL21(DE3) cells transformed with the pGP4
D2D3
expression plasmid was inoculated into 1 liter of Luria-Bertani (LB)
medium containing 60 µg/ml kanamycin and was shaken at 37 °C for
about 6 h until the culture reached an
A600 of ~2.0. The culture flasks were
then chilled on ice for 15 min;
isopropyl-
-D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the induced cultures were
incubated with shaking at 10 °C for an additional 18 h. The
cells did not grow during incubation at 10 °C, yet the gp4
D2D3
protein was expressed as a mixture of soluble and insoluble protein.
The soluble protein accounted for ~10% of the total gp4
D2D3
produced. Protein expression at 22 or 37 °C resulted in higher
levels of gp4
D2D3 expression, but all of the protein was insoluble
in native buffers. After overnight induction at 10 °C, the cells (25 g wet weight) were collected by centrifugation and resuspended in 120 ml of lysis buffer (100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM DTT, and
0.1 mM phenylmethylsulfonyl fluoride). The suspended cells
were lysed by sonication. The cell lysate was centrifuged at
49,000 × g for 40 min at 4 °C, and the supernatant
(fraction I) was collected for purification of gp4
D2D3. About 90%
of the gp4
D2D3 precipitated in the cell lysate, and no attempt was
made to resuspend this insoluble material. 7.7 g of ammonium
sulfate was added to 145 ml of fraction I (10% saturation), and the
solution was centrifuged at 40,000 × g for 40 min at
4 °C. To the supernatant, ammonium sulfate (24.5 g, 40% saturation)
was again added with stirring, and the solution was centrifuged as
before. The resulting pellet containing the gp4
D2D3 protein
(fraction II) was resuspended in 100 ml of Buffer A (20 mM
Tris-HCl (pH 8.0), 0.5 mM EDTA, and 0.5 mM DTT)
and again centrifuged at 49,000 × g for 20 min at 4 °C to remove insoluble material. The clarified fraction II was loaded on a DEAE-Sepharose column (4.9 cm2 × 15 cm) and
washed with 100 ml of Buffer A. The DEAE column was then eluted with a
400-ml gradient of NaCl (0-400 mM) in Buffer A. The
fractions containing gp4
D2D3 were identified by SDS-PAGE and
Coomassie Blue G-250 staining and then pooled (72 ml, fraction III).
Fraction III was diluted to 180 ml with Buffer A to decrease the salt
concentration, and then it was applied in 2 equal aliquots to a Mono Q
column (0.79 cm2 × 10 cm, Amersham Pharmacia Biotech). The
Mono Q column was washed with 20 ml of Buffer A and then eluted with a
120-ml gradient of NaCl (0-400 mM NaCl) in Buffer A. The
fractions containing gp4
D2D3 were combined from both Mono Q
separations (50 ml, fraction IV), diluted to 200 ml with Buffer A, and
loaded onto two 5-ml Hi-Trap heparin-Sepharose columns (2 cm2 × 2.5 cm; Amersham Pharmacia Biotech) connected in
series. After washing the heparin-Sepharose with 50 ml of Buffer A, the
proteins were eluted with a 120-ml gradient of NaCl (0-400
mM) in Buffer A. The fractions containing gp4
D2D3 were
combined, and the protein was precipitated by adding ammonium sulfate
to 60% saturation and then resuspended in 2 ml of Buffer A (fraction
V). Fraction V was loaded onto a Superdex 200 gel filtration column
(5.3 cm2 × 60 cm; Amersham Pharmacia Biotech) that had
been equilibrated with Buffer A containing 100 mM NaCl.
gp4
D2D3 eluted from the gel filtration column at the position of a
66-kDa protein standard, consistent with the monomeric protein. The
purified fractions containing gp4
D2D3 were combined (45 ml, fraction
VI) and concentrated by ultrafiltration (Centriprep; Amicon Inc.) to a
protein concentration of ~20 mg/ml in Buffer A plus 100 mM NaCl. gp4
D2D3 purified from the soluble fraction of
the cell lysate shows no signs of aggregation. The concentrated protein
was used immediately or diluted 2-fold with glycerol (50% v/v final
concentration) and stored at
20 °C. gp4
D2D3 prepared by this
procedure is typically more than 95% pure, as judged by Coomassie Blue
G-250 staining of the protein sample after SDS-PAGE. The
purification procedure typically results in a yield of 2 mg of pure
gp4
D2D3 per liter of culture.
,
-methylene ATP (AMP-PCP, to stabilize the
hexamer), and 40% glycerol were incubated on ice for 30 min before
loading on the gel and electrophoresing the samples for 4 h at 150 V at an ambient temperature of 4 °C. The proteins were visualized
after electrophoresis by staining the gel with Coomassie Blue
G-250.
D2D3. The hydrolysis reactions were incubated for 15 min at
30 °C and stopped by adding 5 µl of 500 mM EDTA. One
microliter of the reaction was spotted on a cellulose polyethyleneimine
plate (J. T. Baker Inc.), which was subsequently developed with 1 M formic acid and 0.8 M LiCl for 1 h.
After drying the gel, the separated substrate and products were
visualized by autoradiography and quantitated by densitometry.
-32P]dGMP into DNA.
The DNA replication reaction (25 µl) contained 40 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 100 µg/ml BSA, 50 mM potassium
glutamate, 0.6 mM each of dATP, dCTP, dGTP, and dTTP, with
333 mCi/mmol [
-32P]dGTP, 100 nM DNA
template, 80 nM T7 DNA polymerase, and the indicated
concentrations of the T7 primase-helicase or gp4
D2D3. The reaction
mixture was incubated at 30 °C, and 4-µl aliquots were removed at
1-min intervals and quenched immediately by the addition of EDTA. The
amounts of DNA synthesized were measured by spotting the reaction
aliquots onto DE81 filters, washing the filters 3 times with 0.3 M ammonium formate (pH 8.0), and measuring the bound
radioactivity by scintillation counting.
-32P]ATP and CTP, and 100 nM T7 primase-helicase or gp4
D2D3. The reaction mixtures
were incubated at 25 °C for 30 min and stopped by the addition of 2 µl of 98% formamide, 20 mM EDTA, 0.1% bromphenol blue,
0.1% xylene cyanol. The products were separated by electrophoresis in
a 25% acrylamide gel containing 3 M urea and visualized by autoradiography.
T7 DNA polymerase, and the
indicated concentrations of the primase-helicase or gp4
D2D3. The
mixtures were incubated for 30 min at 37 °C, and then DNA synthesis
was quantitated as the amount of radioactive dTMP incorporated into DNA
using the DE81 filter binding described above.
D2D3 and T7 DNA polymerase
bound to a primed DNA template using an exonuclease protection assay
(20, 45). DNA complexed to the primase-helicase and the DNA polymerase
is protected from degradation by exonuclease III, whereas the DNA that
dissociates from the complex is rapidly degraded. The assay consists of
a reaction in which a radiolabeled primer is synthesized by the T7
primase and elongated by T7 DNA polymerase, followed by dilution of the
reaction and the re-addition of one or both T7 protein(s) prior to the
addition of a large molar excess of exonuclease III (refer to Fig.
6a). The conditions for synthesis of the radiolabeled primer
are similar to those described above for RNA-primed synthesis of DNA.
The 25-mer DNA template for this reaction,
5'-CAGTGACGGGTCGTTTATCGTCGGC-3' (template 3 in Table IV),
includes a primase recognition site (underlined) for synthesis of the
tetraribonucleotide pppACCC by the primase. The template sequence for
synthesis of the tetraribonucleotide by the primase is followed by a
sequence that allows primer extension by T7 DNA polymerase to be
terminated site-specifically, after the incorporation of 1-4
deoxynucleotides. For example, a 6-mer primer strand (pppACCCdGddT) was
synthesized in a 10-µl reaction containing 40 mM Tris-HCl
(pH 7.5), 10 mM MgCl2, 10 mM DTT,
100 µg/ml BSA, 50 mM potassium glutamate, 0.5 mM template DNA, 1 mM each of
[
-32P]ATP and CTP, 0.3 mM each of dGTP and
ddTTP, and 100 nM each of gp4
D2D3 and exo
T7 DNA polymerase. The primer synthesis reaction was incubated at
25 °C for 30 min, and then it was diluted 100-fold before adding back one or both of the replication proteins (10 µM) and
challenging with exonuclease III. By diluting the initial reaction
mixture to 1 nM protein, we could then add back 10 µM gp4
D2D3 and/or 10 µM T7 DNA
polymerase to determine if both proteins are required for the formation
of a stable priming complex. A high concentration (10 mM)
of the next 2'-deoxynucleotide matching the template was added to
stabilize the 3' end of the primer in the polymerase active site (20).
The final reaction for exonuclease digestion (30 µl) consisted of 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
10 mM DTT, 100 µg/ml BSA, 50 mM potassium
glutamate, 10 mM dCTP, 10 µM each gp4
D2D3
and/or exo
T7 DNA polymerase, and a 100-fold dilution of
the initial primer synthesis reaction (final concentration of the
template DNA was 5 µM). A 4-µl aliquot was taken before
the addition of exonuclease III, and the sample was quenched by
addition of 3 µl of formamide with bromphenol blue. This sample is
regarded as the 0 min time point of exonuclease III digestion. The
nuclease digestion was initiated by adding exonuclease III to the
reaction (4 units/µl; New England Biolabs). The reaction was
incubated at room temperature, and aliquots (4 µl) were removed at
the indicated times and quenched with formamide/dye solution (3 µl).
The radiolabeled products were separated by electrophoresis in a 25%
acrylamide gel containing 3 M urea and then visualized by
autoradiography. Exonuclease III efficiently removes deoxynucleotides
from the 3' end of the radiolabeled primer strand of the unprotected
DNA, leaving a tetraribonucleotide (pppACCC) that is resistant to
further degradation. The time course of DNA dissociation from the
priming complex was determined from the intensity ratio of the fully
extended primer strand at each time point to the intensity at 0 min.
Under these conditions the amount of exonuclease III was not completely
saturating. The addition of 3-fold more exonuclease decreased the
apparent lifetime of the priming complex by about 10-20%. The
continued synthesis of tetraribonucleotides during exonuclease
digestion presents another complication (described below) in
determining the half-life of the priming complex. We have therefore
operationally defined the apparent half-lives of the various priming
complexes (Tables I-V) as the time of exonuclease digestion required
to decrease the amount of extended primer strand to one-half of the
amount present at 0 min.
-32P]dGTP to radiolabel the primer strand. After
primer synthesis, exonuclease challenge was carried out as described
above. pppAC was prepared by enzymatic synthesis using the primase
fragment (residues 1-271) of the primase-helicase (18) and a short DNA template (5'- GTCAA-3'). The reaction (2 ml) contained 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
10 mM DTT, 100 µg/ml BSA, 50 mM potassium glutamate, 2 µM template DNA, 2 mM each ATP
and CTP, and 1 µM T7 primase fragment. The reaction was
incubated for 1 h at 37 °C, and the products were
purified by reverse phase high performance chromatography
(Delta Pack C18 300 Å, Waters). The molecular mass of the
purified pppAC was confirmed by matrix-assisted laser desorption ionization/time of flight mass spectrometry (theoretical mass, 812.4;
measured mass, 813.2).
D2D3 and
exo
T7 DNA polymerase (final concentration of the
template DNA was 10 µM in the binding reaction). The
binding mixture was incubated on ice for 30 min, and the avidin resin
was washed three times with 200 µl of wash buffer at 4 °C using a
centrifugal filtration device (Nanosep MF; Pall Filtron). The wash
buffer consisted of 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 50 mM potassium glutamate, 0.2% Tween 20, 200 mM
NaCl, and 5% glycerol. The Tween 20 detergent was added to prevent
nonspecific binding of proteins to the filtration membrane. 10 mM dCTP was added to the wash solution of complexes that
were initially prepared in the presence of dCTP. The washed avidin
beads were resuspended in 20 µl of SDS-PAGE sample buffer and
centrifuged to elute the bound proteins. The eluted proteins were
separated by SDS-PAGE and visualized by staining with Coomassie Blue
G-250.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix of
the helicase domain (helix A; residues 272-281; Fig. 1b) extends from each subunit
to pack against three helices of the adjacent subunit (helices D1-D3;
residues 345-388) in a "helix swapping" arrangement (46). The
hexamer is further stabilized by additional residues immediately
N-terminal to helix A (19, 30) and several loops surrounding the
nucleotide-binding site. Because the isolated primase domain is
monomeric and shows no evidence of self-association even at high
protein concentrations (19), it seemed likely that the main
interactions stabilizing the primase-helicase hexamer were those
observed in crystal structures of the helicase domain.
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Fig. 1.
Domain organization of T7 gene 4 protein. a, the modified region of the gp4 D2D3
primase is shown (boxed region) along with the corresponding
sequence of the primase-helicase. A linker between the primase domain
and the helicase domain (residues 245-272) is susceptible to
proteolysis (19). The secondary structure of the helicase domain (29,
30) is shown above the conserved sequence motifs, with
boxes denoting
-helices and arrows for
-strands. Helices D2 and D3 (residues 368-382) within the helicase
domain are replaced in gp4
D2D3 with the polar segment SASASG. The
boundaries and conserved motifs of the primase and helicase domains are
shown, as defined by Ilyina et al. (55). b, two
subunits of the hexamer are shown, colored green and
orange, respectively. The interface between subunits of the
primase-helicase includes the active site of the helicase (with bound
dATP) and the swapped helix A that packs against helices D1-D3 of the
neighboring subunit (29, 30).
D2D3 to indicate the deletion of helices D2 and D3, lacks half
the residues contributing to the helix swapping interaction that links
adjacent subunits of the primase-helicase. Although gp4
D2D3 is
insoluble when expressed in E. coli grown at 37 °C, a
significant amount of soluble protein can be obtained by inducing
protein expression at 10 °C for 18 h (Fig.
2a; see "Experimental
Procedures"). The gp4
D2D3 purified from the soluble fraction is
well behaved and completely soluble at 25 mg/ml in buffer containing
100 mM NaCl. About 18 mg of soluble protein can be obtained
from a 9-liter culture.
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Fig. 2.
Purification of the
gp4 D2D3 primase. a, the
purification scheme of the soluble gp4
D2D3 is described in detail
under "Experimental Procedures" and is summarized by the gel
analysis of the column fractions shown here. Lanes MW,
molecular weight markers showing their molecular mass to the
left of the gel; Supernatant, the supernatant of
the cell lysate; Pellet, the precipitate of the cell lysate;
40% (NH4)2SO4, the
pellet of 40% ammonium sulfate precipitation; DEAE, the
pool of fractions from the DEAE column; Mono Q, the pooled
fractions after the Mono Q column; Heparin-Sepharose, the
pool of fractions of the Hi-trap Heparin column; Superdex
S200, the collected fractions from the gel filtration column.
b, T7 primase-helicase and gp4
D2D3 were analyzed on a
15% native gel. The sample buffer contained 5 mM
,
-methylene ATP, and the electrophoresis buffer contained 1 mM ATP to stabilize oligomers of the proteins (10, 11). The
positions of molecular weight markers are indicated. During
electrophoresis, the wild-type primase-helicase (10 µM)
migrates with an apparent mass of 380 kDa (marked with an
asterisk), consistent with the formation of a protein
hexamer. A slowly migrating species is also evident, and it might
consist of two hexamers stacked together. gp4
D2D3 (10 µM) predominantly migrates as a monomer (
70 kDa).
Although several gp4
D2D3 oligomers are also present in the gel, no
hexamers are detected.
D2D3 was examined by native gel
electrophoresis (Fig. 2b). 5 mM
,
-methylene ATP and 1 mM ATP was included in the
protein sample and in the gel running buffer, respectively, in order to
favor the formation of oligomers (11, 47). Nucleotides bind at the
interfaces between subunits of the primase-helicase (29, 30) and
stabilize the hexameric form of the protein (10). The T7
primase-helicase has an apparent mass of ~400 kDa, indicating that
the hexamer forms under these conditions (Fig. 2b). Variable
amounts of a second, slowly migrating species are observed in current
and previous experiments (11, 48). The apparent mass of this slowly
migrating species is consistent with two hexamers associated in the low
ionic strength buffer used for native PAGE. In contrast to the
wild-type primase-helicase, about 85% of the gp4
D2D3 is monomeric
(
70 kDa) in the gel, and there is no detectable hexamer.
However, trace amounts of several higher order oligomers of gp4
D2D3
are evident in the native gel, indicating some self-association into
dimers and trimers.
D2D3 Lacks dTTPase and DNA Unwinding
Activities--
Although the gp4
D2D3 is predominantly monomeric in
solution, it was important to determine whether the modified protein
can assemble into functional hexamers on DNA. We therefore assayed gp4
D2D3 for its ability to hydrolyze dTTP and to unwind DNA, activities that depend upon the oligomerization of gp4. The hydrolysis of dTTP fuels the translocation of the primase-helicase along DNA in a
5' to 3' direction, separating the strands of duplex DNA (22-26). The
active site of the helicase consists of residues from two adjacent
subunits, and oligomerization is required for nucleotide hydrolysis
(19). The addition of single-stranded DNA to the primase-helicase
stimulates its rate of dTTP hydrolysis about 20-fold and promotes
hexamer formation (11, 47). In contrast, gp4
D2D3 lacks detectable
dTTPase activity in the presence or absence of M13 ssDNA (not shown),
even at protein concentrations as high as 50 µM using
conditions that support a high level of nucleotide hydrolysis activity
by the wild-type primase-helicase. The results indicate that gp4
D2D3
does not form functional hexamers, even at protein concentrations that
are much higher than those required to assemble a hexamer of the
wild-type primase-helicase (Fig. 2b).
D2D3 also lacks DNA unwinding activity and thus is unable to
assemble on DNA into functional hexamers (Fig.
3). The DNA unwinding activity of the
wild-type primase-helicase readily displaces a radiolabeled
oligonucleotide annealed to M13 ssDNA, whereas gp4
D2D3 is inactive,
even at 100-fold higher protein concentrations (Fig. 3). Helicase
activity was also assayed in a rolling circle DNA replication system in
which DNA strand separation by the helicase is required for DNA
synthesis (Fig. 4) (2). In the presence of the primase-helicase, T7 DNA polymerase incorporates radiolabeled nucleotides into DNA at a rate of 2-3 pmol/min. In contrast,
gp4
D2D3 does not support DNA synthesis (incorporation of <0.1
pmol/min) in this replication system even at 10 µM
gp4
D2D3, consistent with its lack of DNA unwinding activity. We
could not exclude the possibility that gp4
D2D3 is inactive in this
replication system because it fails to interact with the DNA or
proteins of the replication fork (2, 49). We therefore examined
whether or not gp4
D2D3 could inhibit the activity of wild-type
primase-helicase in this reaction. When gp4
D2D3 (10 µM) was mixed with the primase-helicase (60 nM) in the replication reaction, the rate of DNA synthesis was decreased to 1/2-
D2D3 can interact with the T7 DNA replication
complex and interfere with DNA synthesis, presumably because the
altered protein cannot unwind the DNA duplex.
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Fig. 3.
gp4 D2D3 lacks DNA
unwinding activity. The DNA unwinding activity of the
primase-helicase was measured by the displacement of a radiolabeled
37-mer oligonucleotide annealed to M13 ssDNA. After incubation with
gene 4 proteins, the substrates were separated by electrophoresis
through a native gel (15%) and visualized by autoradiography.
Lane 1 shows the 5'-32P-labeled oligonucleotide
alone, and lane 2 shows the starting substrate with the
oligonucleotide annealed to M13 ssDNA. The primase-helicase has
significant DNA unwinding activity, assayed at several different
protein concentrations in the presence of dTTP (lanes 3 and
4; see "Experimental Procedures" for details).
gp4
D2D3 lacks detectable DNA unwinding activity even at 10-fold
higher protein concentration (lanes 5-7).
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Fig. 4.
gp4 D2D3 does not
support synthesis of DNA catalyzed by T7 DNA polymerase on duplex
templates. A rolling circle DNA replication reaction (2) was used
to determine if gp4
D2D3 supports the replication of double-stranded
DNA. The reaction contained 0.6 mM each of dATP, dCTP,
dGTP, and dTTP with 333 mCi/mmol [
-32P]dGTP, 100 nM DNA template (see inset), 80 nM
T7 DNA polymerase, and the indicated concentrations of the
primase-helicase or gp4
D2D3. The incorporation of
[
-32P] dGMP into DNA was monitored (see
"Experimental Procedures" for the details). The primase-helicase
functions in the efficient replication of a 70-nucleotide
(70-nt) circular DNA, whereas gp4
D2D3 does not,
apparently because it lacks DNA unwinding activity even within the
context of the replication protein complex.
D2D3 Primes DNA Synthesis by T7 DNA Polymerase--
The
synthesis of RNA primers by the primase-helicase and by gp4
D2D3 was
examined in reactions with a 25-mer DNA template containing a primase
recognition site (template 3 in Table IV). In the presence of
[
-32P]ATP and CTP, gp4
D2D3 synthesized the same
amounts of di-, tri-, and tetraribonucleotide products as the wild-type
primase-helicase (Fig. 5a).
The primase-helicase and gp4
D2D3 also synthesize a small amount of a
pentaribonucleotide, presumably by misincorporating AMP or CMP opposite
a cytidine at this position of the template. The major product of RNA
synthesis is a tetraribonucleotide that can be elongated by T7 DNA
polymerase in the presence of dNTPs (see Fig.
6b).
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Fig. 5.
Synthesis of RNA primers and utilization for
DNA synthesis. a, the primase activities of the
primase-helicase and gp4 D2D3 mainly synthesize a tetraribonucleotide
that can be utilized by T7 DNA polymerase and lesser amounts of a pppAC
dinucleotide that cannot be elongated by the polymerase (15). The
incorporation of [
-32P]ATP into oligoribonucleotides
by T7 primase was analyzed in reactions containing 0.1 mM
25-mer template (template 3 in Table IV), 1 mM each of
[
-32P]ATP and CTP, and 100 nM T7
primase-helicase or gp4
D2D3. The products of this reaction were
separated by electrophoresis in a 25% polyacrylamide gel containing 3 M urea and visualized by autoradiography. Both primases
produce similar amounts of the RNA products shown. b, the
synthesis of tetraribonucleotides by the primase-helicase and their
elongation by T7 DNA polymerase were carried in the reactions
containing 25 ng/µl M13 ssDNA, 1 mM each of ATP and CTP,
0.3 mM each of dATP, dCTP, and dGTP, 0.3 mM
[3H]dTTP, 100 nM of exo
T7 DNA
polymerase, and the indicated concentrations of the primase-helicase or
gp4
D2D3. After incubation at 37 °C for 10 min, incorporation of
[3H]dTTP into DNA was measured by scintillation counting.
Like the wild-type primase-helicase, gp4
D2D3 catalyzes the
RNA-primed synthesis of DNA on a M13 ssDNA template. The gp4
D2D3
protein, which is predominantly monomeric and lacks the dTTPase
activity associated with the primase-helicase, does not inhibit DNA
synthesis at high concentrations, and it supports DNA synthesis at a
rate that is about 1/10th the maximal rate of the wild-type
primase-helicase in this assay.
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Fig. 6.
Formation of a long lived priming
complex. a, a stable complex of T7 DNA polymerase and
gp4 D2D3 bound to a primed DNA template can be assembled under
conditions that block extension of the primer strand by the polymerase.
The primase synthesizes a tetraribonucleotide (red) that can
be extended by T7 DNA polymerase. DNA synthesis is terminated by the
incorporation of a 2',3'-dideoxynucleotide. The primer synthesis
reaction is then diluted 100-fold so that the priming complex can be
assembled on the primed template using one or both T7 replication
proteins. The protein-DNA complex protects the radiolabeled primer
strand from degradation by exonuclease III. The addition of dCTP, which
binds to the polymerase active site, secures the polymerase to the 3'
end of the primer strand (45). dCTP is not present during primer
synthesis, so the polymerase and primase readily dissociate from the
primed DNA template (step 1), prior to assembly of the
stable priming complex in step 2. The stability of this
primer elongation complex is measured by challenging the protein-DNA
complex with exonuclease III and monitoring the degradation of the
radiolabeled primer strand as the complex dissociates. Exonuclease III
removes the deoxynucleotides from the 3' end of the primer, leaving
a tetraribonucleotide (pppACCC) that is resistant to further
degradation. b, an autoradiograph showing the radiolabeled
products of the primer extension reaction described in a,
after challenge with exonuclease III. The exonuclease challenge assays
were carried out as described under "Experimental
Procedures," and the degradation of the radiolabeled product
(pppACCCdGddT) at the indicated times was analyzed by
electrophoresis in a 25% native gel and visualized by autoradiography.
The lengths of the radiolabeled products are shown at the left
side of the figure, including the triribonucleotide (pppACC;
labeled 3) and the tetraribonucleotide (pppACCC; labeled
4) synthesized by the primase. The primer extension products
resulting from the incorporation of deoxynucleotides by T7 DNA
polymerase include a 5-mer (pppACCCdG) and a specifically terminated
6-mer product (pppACCCdGddT). In reaction 1, the addition of
both T7 replication proteins (10 µM each) and a bound
nucleotide substrate (dCTP) results in a very stable protein-DNA
complex with a half-life of more than 60 min. Omitting any of these
components (reactions 2-5) from the binding reaction
results in the loss of protection of the radiolabeled strand.
c, the intensities of the 6-mer primer extension products of
reactions 1-5 shown in b are plotted as a fraction of the
starting material at 0 min for each reaction. The continued synthesis
of new primer strands with residual nucleotide substrate is a likely
explanation for the initial increase in product abundance
(reaction 1) during exposure to exonuclease III.
D2D3 are both predominantly monomeric proteins, and primer utilization by T7 DNA polymerase might require the presence of a
hexameric primase. We tested this notion by measuring the amount of DNA
synthesis catalyzed by T7 DNA polymerase in reactions containing the
hexameric T7 primase-helicase or the predominantly monomeric gp4
D2D3
protein. As expected, the wild-type primase-helicase supported the
RNA-primed synthesis of DNA by T7 DNA polymerase in a reaction
containing an M13 ssDNA template with ATP, CTP, and a mixture of all
four dNTPs. The rate of DNA synthesis increased with increasing
concentrations of the primase-helicase protein until DNA synthesis was
inhibited at very high protein concentrations (Fig. 5b).
This inhibition of DNA synthesis in the presence of a large molar
excess of primase-helicase probably results from the depletion of dTTP
in the DNA synthesis reaction through hydrolysis by the helicase
domain. Remarkably, gp4
D2D3 supports DNA synthesis at a rate
approaching 1/10th the wild-type rate. High concentrations of
gp4
D2D3 do not inhibit DNA synthesis, a finding consistent with its lack of dTTPase activity. The lower efficiency of gp4
D2D3 in promoting primer utilization and DNA synthesis might be explained by
its failure to rapidly locate priming sites on the M13 ssDNA because of
the defective helicase domain, which is unable to actively translocate
on DNA (11).
D2D3 and T7 DNA
Polymerase--
Although it is incapable of assembling into functional
hexamers, gp4
D2D3 primes DNA synthesis catalyzed by T7 DNA
polymerase. This finding suggests that a single subunit within the
hexameric primase-helicase is sufficient to prime Okazaki fragment
synthesis on the lagging strand of the replication fork. The
requirement for the primase during extension of RNA primers further
suggests that the T7 DNA polymerase engages short, naturally occurring primers only when they are bound to the primase protein. The primase might help initiate primer elongation by securing the RNA primer to the
DNA template (3) and/or by recruiting the polymerase to the priming
site through protein-protein interactions. A four amino acid loop
located at the base of the thumb of T7 DNA polymerase is required for
lagging strand synthesis (20). The selective loss of lagging strand
synthesis upon deletion of the loop, together with its location near
the polymerase active site, suggests that the loop might contact the
RNA primer or interact with the primase during the elongation of a primer.
D2D3 primase to synthesize and deliver tetraribonucleotide primers to the polymerase. A very stable
protein-DNA complex containing gp4
D2D3 and T7 DNA polymerase is
formed when DNA synthesis is halted by the incorporation of a
2',3'-dideoxynucleotide during the initial cycles of deoxynucleotide
incorporation by the polymerase (Fig. 6a). The stability of
the protein-DNA complex was determined by adding a high concentration
of exonuclease III and monitoring the loss of the radiolabeled primer
as DNA dissociates from the complex (Fig. 6a). Both
gp4
D2D3 and T7 DNA polymerase are required for the formation of the
stable complex with the nascent primer, suggesting that both proteins
remain bound to DNA (compare the extent of protection for
reaction 1 with that of reactions 3-5 in Fig.
6b). If either protein is left out of the binding reaction,
the primer is rapidly degraded to a tetraribonucleotide (labeled at the
5'
-phosphate position), which resists further degradation by
exonuclease III. A high concentration of the substrate dCTP is also
required to secure the primer in the active site of the polymerase
(reaction 2, Fig. 6b) (20, 45). In the complete reaction, the radiolabeled primer DNA strand is very resistant to
degradation by exonuclease III, and the protein-DNA complex dissociates
slowly (Fig. 6c). The exposed 3' single-stranded end of the
DNA template is a poor substrate for exonuclease III (50-52), and it
is degraded slowly (results not shown) in comparison to the recessed 3'
end of the primer. A determination of the lifetime of the protein-DNA
complex is complicated by an observed 2-fold increase in the amount of
radiolabeled primer synthesized during the first 15 min of the
exonuclease digestion (compare reaction 1 with
reactions 3-5, Fig. 6c). This increase is the
result of de novo primer synthesis by the primase (10 µM) present during exonuclease digestion. Because of
these complications, we operationally define the apparent half-life of
the priming complex as the time when the amount of extended primer
decreases to one-half the amount at 0 min. The apparent
half-lives of priming complexes measured in this way (Tables
I-V) can then be compared to identify
the experimental variables that affect complex stability. The priming
complexes containing the gp4
D2D3 monomer are almost as long lived as
the priming complex containing the hexameric T7 primase-helicase
(20).
The apparent lifetime of the priming complex depends upon the
concentration of the bound nucleotide substrate
D2D3 is
not required for the formation of the priming complex. A truncated
version of gp4
D2D3 lacking this acidic segment forms priming
complexes with T7 DNA polymerase that are as stable as those formed by
the full-length gp4
D2D3 protein described above (not shown). The
lack of involvement of the C-terminal segment of the helicase domain
further implies that the priming complex is sustained by interactions
between the primase domain of the primase-helicase and the polymerase.
At very high protein concentrations (100 µM primase), the
primase fragment (residues 1-271 (18)) of the primase-helicase lacking
the helicase domain can function in the assembly of a short lived
priming complex (half-life 1-2 min determined by exonuclease
challenge; not shown). The added stability provided by the helicase
domain of gp4
D2D3 might result from tighter interactions with the
DNA template (40).
-32P]dGTP at the 3' end of the
tetraribonucleotide primer followed by the incorporation of ddTMP to
terminate DNA synthesis. The addition of dCTP, which matches the next
position of the template, produced a long lived priming complex,
whereas the addition of dGTP resulted in a half-life of less than 5 min
(Fig. 8 and Table II). The complex with
dATP was initially degraded rapidly by the added exonuclease, but some
of the radiolabeled primer was protected from degradation for more than
2 h. Surprisingly, a pairing of dTTP with the template guanosine
was fairly effective at stabilizing the priming complex (Fig. 8). It is
possible that the 10 mM dTTP present in the reaction
permits re-synthesis of the extended primer following the
exonucleolytic removal of the 3'-ddTMP.
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Fig. 7.
Stabilization of the priming complex by the
polymerase substrate dCTP. A high concentration of the next
nucleotide (dCTP) matching the template sequence stabilizes the priming
complex. The exonuclease III protection assay shown in Fig. 6 was used
to monitor the formation of the priming complex in the presence of the
nucleotide concentrations shown. Nucleotide concentrations higher than
5 mM (not shown) did not additionally increase the lifetime
of the priming complex.
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Fig. 8.
Mismatched dNTPs can stabilize the priming
complex. The stabilities of priming complexes assembled with the
correct (dCTP) or mismatched nucleotide substrates were measured as
described in the legend of Fig. 6, using 10 mM of each of
the nucleotides listed above the gel. The extended primer
strand was labeled by the incorporation of [ -32P]dGMP
into DNA, followed by chain termination with 2',3'-dideoxythymidine.
The sequences of the extended primer strands are indicated on the
left side of the figure.
The correct bound nucleotide, and some incorrect nucleotides,
stabilize the priming complex
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Fig. 9.
The stability of the priming complex changes
during elongation of the primer strand. The primer extension
reaction (described in Fig. 6) was stopped at different positions of
the DNA template by the incorporation of a chain-terminating
2',3'-dideoxynucleotide. After the addition of gp4 D2D3, T7 DNA
polymerase, and the appropriate dNTP, the stabilities of the resulting
priming complexes were determined by exonuclease challenge. The complex
containing the 6-mer primer extension product (pppACCCdGddT) is the
most stable. Incorporation of fewer or more nucleotides prior to
terminating synthesis resulted in faster dissociation of the
protein-DNA complex (5-, 7-, and 8-mer complexes). The effect of primer
strand length on the stability of the protein-DNA complex implies that
specific contacts are made to the 5' and 3' ends of the primer strand
within the complex.
The stability of the priming complex depends upon the length of the
primer strand in the complex
D2D3 and their
elongation by T7 DNA polymerase (data not shown). However, the
stabilities of the stalled priming complexes depended upon template
length (Table IV). The complex with the shortest template (a 19-mer, template 1 in Table IV) was least stable and had a half-life of less
than 5 min. Addition of three bases to the 3' end of this sequence
(template 2) significantly improved the stability of the priming
complex (Table IV). A three-nucleotide extension to the 5' end of
template 2 to create template 3 provided optimal stability. A template
with three additional nucleotides at the 3' end (template 4) produced
an equally stable priming complex. However, the addition of nucleotides
to the 5' end of the template (templates 5 and 6 in Table IV)
destabilized the complex. The reason for this effect is not known.
DNA template requirements for the T7 priming complex
D2D3 and T7 DNA polymerase in
primer extension reactions initiated with pppAC or AC dinucleotides
instead of ATP. Both dinucleotides were readily extended by gp4
D2D3,
and the resulting tetraribonucleotide was elongated by T7 DNA
polymerase in the presence dNTP and ddNTP substrates (Fig.
10). The priming complex that was
initiated with pppAC was as long lived as the complex initiated with
ATP. In contrast, the priming complex initiated with the
unphosphorylated AC was unstable, with a half-life of less than 5 min
(Fig. 10 and Table V). These results show
that the 5'-triphosphate of the primer strand contributes significantly
to the stability of the priming complex, and they imply that the
phosphate group plays a role in primer utilization.
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Fig. 10.
The 5'-triphosphate of the primer stabilizes
the priming complex. Priming complexes were initiated with a
phosphorylated dinucleotide (pppAC) or with the unphosphorylated
dinucleotide (AC) and elongated by the primase and T7 DNA polymerase in
the presence of [ -32P]dGTP to form radiolabeled
products with the sequences shown in the figure. The stabilities of
priming complexes initiated with either dinucleotide were compared with
that of a standard complex in which primer synthesis was initiated with
ATP (left side of figure), using the exonuclease challenge
assay described in Fig. 6. The unphosphorylated primer strand migrates
more slowly than the phosphorylated strands. It is evident that priming
complexes containing a 5'-triphosphate group on the primer strand
(initiated with pppAC or ATP) are significantly more stable than the
complex with the unphosphorylated primer.
The 5'-triphosphate of the primer contributes to stability of the
priming complex
D2D3 and T7 DNA polymerase remained
associated with the template. If either the primer-template strand or
the dCTP was left out of the reaction, no detectable protein remained
associated with the immobilized template (lanes 3-5). When
the correct incoming nucleotide (dCTP) was replaced with 10 mM dTTP, dATP, or dGTP a stable complex was not isolated
(data not shown), providing further evidence of the specificity of
protein interactions with the immobilized DNA template. Although a
mismatched dTTP provided some protection against exonuclease digestion
(see above), the isolation of the priming complex requires the correct
nucleotide substrate to be present during complex formation and in the
wash buffer. The equal Coomassie Blue staining intensities of the
gp4
D2D3 and T7 DNA polymerase eluted from the avidin-agarose beads
(Fig. 11, lane 2) suggest that a 1:1 complex of primase and
DNA polymerase is present in the stalled priming complex.
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Fig. 11.
Isolation of a stable DNA complex containing
both primase and polymerase. The priming complex described in the
legend of Fig. 6 was purified using an immobilized DNA template that
was attached to avidin-agarose beads through a 3'-biotin group. After
washing the beads, the proteins were eluted with SDS and separated by
electrophoresis on a 12% SDS-PAGE gel and then visualized by staining
with Coomassie Blue. The priming complex (lane 2) contains
both T7 DNA polymerase (the purified protein is shown in lane
6) and gp4 D2D3 (compare with pure protein in lane
7). The equal staining intensities of the proteins eluted from the
priming complex suggest that T7 DNA polymerase and gp4
D2D3 are
present in a 1:1 ratio. Neither protein alone stably interacts with the
immobilized DNA (not shown). Immobilization of the proteins on the
agarose beads requires dCTP (lane 3) and the DNA template
(lanes 4 and 5), indicating that a specific
complex forms on the primed DNA template.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
D2D3) that
does not form functional hexamers, yet it primes DNA synthesis by T7
DNA polymerase (Fig. 5b). The monomeric primase physically complexes with T7 DNA polymerase during initiation of RNA primer extension. These results suggest that the primase complexed to the DNA
template delivers the newly synthesized tetraribonucleotide primer to
the polymerase active site. Although gp4
D2D3 lacks two
-helices
(D2 and D3) that account for most of the buried surface of the subunit
interface of the helicase domain (Fig. 1b) (29, 30), the
modified protein has some residual tendency to oligomerize (Fig.
2b). The remaining interactions between subunits might be
mediated by the N-terminal primase domain or by the loops surrounding
the active site of the helicase (29, 30). Additionally, the linker
region (residue 241-271) between the primase and helicase domain
contributes to oligomerization of the gene 4 protein (19). gp4
D2D3 lacks the dTTPase and DNA unwinding activities associated with the hexameric primase-helicase, and it is predominantly monomeric, even at high protein concentrations (Fig. 2b). These results
strongly suggest that a single protomer of the T7 primase-helicase can function in the utilization of tetraribonucleotide primers by T7 DNA polymerase.
D2D3 consists of both primase and helicase domains of the T7
gene 4 protein. A fragment of the primase-helicase spanning only the primase domain is a monomer, and it catalyzes the
template-dependent synthesis of tetraribonucleotides at
specific priming sites (18), as does gp4
D2D3. However, the minimal
primase fragment does not efficiently function in primer utilization by
T7 DNA polymerase. The additional helicase domain of gp4
D2D3
might support primer utilization by contributing DNA interactions that
secure the nascent primer on the DNA template until it is elongated by
T7 DNA polymerase (40). The helicase domain might also participate in
protein-protein interactions with the polymerase. The primase-helicase
interacts strongly with T7 DNA polymerase in the absence of DNA (3, 4, 19, 53). However, the priming complex (Ref. 20 and this work) is
different from a previously described interaction between the
primase-helicase and T7 DNA polymerase that involves 17 residues at the
C terminus of the helicase domain (4). This protein-protein interaction
is required to couple the synthesis of the lagging strand of the
replication fork with synthesis of the leading strand (2). This
interaction, which can be observed with the purified proteins bound to
single-stranded DNA (4), might correspond to the interaction of the
helicase on the lagging strand of the replication fork with the
polymerase on the leading strand. In contrast, the interaction of the
T7 primase with the DNA polymerase occurs under conditions specific for
primer elongation. The formation of a stable complex requires a primed
template and a nucleotide substrate bound to the polymerase (Figs. 6
and 11). The removal of the acidic segment from the C terminus of
gp4
D2D3 does not interfere with the formation of a stable priming
complex. These results do not exclude the possibility of additional
protein-protein interactions between the primase and the polymerase
when these proteins are bound to a primed DNA template.
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ACKNOWLEDGEMENTS |
---|
We thank Shenyuan Guo for the gift of a
truncated gene 4 containing the D2D3 mutation that was
used as a starting point and for inspiring us to pursue this project.
We also appreciate the advice and interest of current members of the
Ellenberger and Richardson research groups.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health (to T. E. and C. C. R.) and the Department of Energy (to S. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This manuscript is dedicated to the memory of Shenyuan Guo.
Supported by the postdoctoral fellowships for research abroad from
the Japan Society for the Promotion of Science.
§ To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0458; Fax: 617-432-3380; E-mail: tome@hms.harvard.edu.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M101470200
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ABBREVIATIONS |
---|
The abbreviations used are:
ssDNA, single-stranded DNA;
DTT, dithiothreitol;
AMP-PCP, adenosine
5'-(,
-methylene triphosphate);
PAGE, polyacrylamide gel
electrophoresis;
BSA, bovine serum albumin;
ddTMP, 2',3'-dideoxythymidine.
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