(Received for publication, December 18, 1996)
From the Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115
The 63-kDa gene 4 primase of bacteriophage T7
recognizes a core trinucleotide sequence, 5-GTC-3
, on single-stranded
DNA at which it catalyzes the synthesis of the ribodinucleotide pppAC. The dinucleotide is extended to a tetranucleotide primer at the sites
5
-(G/T)GGTC-3
and 5
-GTGTC-3
. In the presence of T7 primase, T7 DNA
polymerase extends the synthetic ribotetranucleotide pACCA (1 µM), but not pCACA, on M13 DNA templates. The
reaction is specific for T7 DNA polymerase and depends on dTTP and
translocation of the gene 4 protein. T7 primase extends the
dinucleotide AC and trinucleotide ACC to ACCC in the presence of CTP
and an appropriate template, whereas other dinucleotides are extended
less efficiently; the deoxyribodinucleotide dAC is not extended. The
Cys4 zinc motif of the primase is essential for extension
of the dinucleotides. The 5
-cryptic cytidine of the recognition
sequence is essential for extension of the dinucleotide AC to tri- and
tetranucleotides. At a preformed replication fork, the
dinucleotide AC provides for primer synthesis on the lagging strand.
The synthesis of all Okazaki fragments is initiated by primers arising
from the recognition sequence 5
-GGGTC-3
; none arise at an adjacent
5
-GGGTT-3
sequence. If ADP or AMP replaces ATP in the primase
reaction, primers terminating in di- or monophosphate, respectively,
are synthesized.
DNA primases catalyze the template-directed synthesis of
oligonucleotides for use as primers by the lagging strand DNA
polymerase. The primases of T7, T4, and Escherichia coli all
initiate oligonucleotide synthesis on single-stranded DNA
(ssDNA)1 at basic trinucleotide recognition
sites unique to each system (1-5). The 3-nucleotide of the
recognition sequence, in each case, is cryptic in that it is essential
for recognition, but is not copied into the product oligonucleotide.
Although the basic recognition sequence is a trinucleotide, with the
potential to generate a dinucleotide, the oligonucleotides that
actually function as primers for the T7, T4, and E. coli
primases are tetra-, penta-, and 10-12-mers, respectively (6-10). The
stringency for these subsequent nucleotide additions is less than that
observed for the first two nucleotides polymerized at the basic
trinucleotide sequence (1, 3, 4, 7, 8, 11).
The molecular mechanism by which prokaryotic DNA primases recognize a trinucleotide sequence on ssDNA is not known. However, the T7, T4, and E. coli primases have a potential metal-binding site, as do all known DNA primases (12, 13); the T7 and E. coli primases have been shown to be zinc metalloproteins (13, 14). In the case of the T7 primase, the Cys4 zinc motif is located at the amino terminus of the protein; the substitution of a serine for any one of the four cysteines destroys its ability to catalyze the synthesis of oligonucleotides in a sequence-specific manner (13). However, altered T7 primases containing these single amino acid changes or even lacking the entire zinc motif still catalyze the DNA-independent formation of random dinucleotides, albeit at a greatly reduced rate, demonstrating that the site for phosphodiester bond formation is located in another domain of the protein (13, 15, 16). The zinc motif, however, is not the sole determinant of primase site recognition since we have shown that substituting the zinc motifs of the T4 and E. coli primases for that of the T7 primase does not lead to the creation of a chimeric primase that now uses the T4 or E. coli recognition sequence, but rather to one that recognizes an entirely new trinucleotide sequence (17).
Although the T7 primase shares many properties with the primases of phage T4 and E. coli, it has a number of distinguishing properties. A unique feature of the T7 DNA primase is the presence of a helicase domain, which allows the 63-kDa gene 4 protein, a protein composed of a single polypeptide chain, to catalyze both helicase and primase activities. (15, 18). In contrast, in the T4 and E. coli systems, helicase and primase activities reside within separate polypeptides (8, 19-22). Physical association of primases with a helicase in a functional complex is important because the translocation activity of the helicase provides a mechanism for the primase to reach its recognition sites on ssDNA (2, 9, 20, 21, 23). The presence of both helicase and primase activities in the same T7 protein arises from the presence of two co-linear gene 4 proteins, a 56- and a 63-kDa protein, in phage-infected cells (24). The two proteins are expressed from two in-frame translation initiation sites 189 bases apart on the gene 4 transcript. The 56-kDa gene 4 protein lacks the 63 N-terminal amino acid residues of the 63-kDa gene 4 protein, and it is this domain that contains the zinc motif essential for primase activity. Thus, the 56-kDa gene 4 protein has only helicase activity, whereas the 63-kDa gene 4 protein has both helicase and primase activities (16, 18). In this report, we refer to the 63-kDa gene 4 protein as DNA primase even though it has helicase activity.
A second distinguishing property of the T7 primase involves the selectivity in the particular nucleotides incorporated into the primers after the invariant dinucleotide is synthesized at the basic primase recognition site. T7 primase greatly prefers AMP and CMP in the third and fourth positions of the primer, whereas the T4 and E. coli enzymes are less restrictive in the nucleotides incorporated (25, 26). We have shown that this specificity is, at least in part, due to the inherent translocation activity of the T7 primase, which does not allow the enzyme to pause for sufficient time to incorporate unfavorable nucleotides (11).
A third distinguishing property, and the subject of this study, is the
ability of the T7 primase to synthesize relatively large amounts of the
dinucleotide pppAC together with the functional primer species, a
tetranucleotide. The actual RNA primers found at the 5-termini of
Okazaki fragments synthesized in cells infected with phage T7 (6) or in
reactions containing the T7 primase and T7 DNA polymerase are
tetranucleotides, predominantly pppACCC, pppACCA, and pppACAC (7,
26). These tetranucleotides arise from the general recognition sites
5
-GGGTC-3
, 5
-TGGTC-3
, and 5
-GTGTC-3
, respectively, all containing
the core recognition sequence for the T7 primase, 5
-GTC-3
(7). On
naturally occurring ssDNA templates such as phage M13 DNA or on
synthetic oligonucleotides containing one of the general recognition
sites, the T7 primase, in the presence or absence of coupled DNA
synthesis, catalyzes the synthesis of di- and trinucleotides as well as
the functional tetranucleotide primers (18). In fact, on templates
containing only the core trinucleotide sequence 5
-GTC-3
, the enzyme
catalyzes exclusively the synthesis of the dinucleotide pppAC (18).
Although small amounts of dinucleotide have been observed with the T4
and E. coli primases under some conditions, their
abundance is far less than that seen with T7 primase (2, 27).
The ability of the T7 primase to catalyze the exclusive synthesis of
dinucleotides at core recognition sites (5-GTC-3
) not contained
within a general recognition site is intriguing and raises the
possibility that dinucleotides may serve some functional role in the
priming reaction. One possibility is that, although the dinucleotides
cannot be extended at these sites, they may remain associated with the
primase during its translocation to a general recognition site, where
extension to a functional primer could occur. In early studies on the
gene 4 protein, Scherzinger et al. (28) reported that
certain tri- and tetranucleotides could stimulate DNA synthesis by T7
DNA polymerase on ssDNA templates in a reaction dependent on the
presence of the T7 gene 4 protein. Interestingly, of the
tetranucleotides tested, ACCA was the most effective, and priming
appeared to be initiated at specific sites on
X174 DNA; in this
study, dinucleotides were without effect. Subsequent studies revealed
that the 63-kDa gene 4 protein alone could stimulate T7 DNA polymerase
in the presence of a tetranucleotide (15). In this report, we show that
the ribodinucleotide AC can be extended to a tetranucleotide by the
63-kDa gene 4 protein and that the reaction occurs only at a general
primase recognition site, 5
-NNGTC-3
, containing the essential but
cryptic cytidine residue. In addition, we examined the use of
dinucleotides in the priming reaction at a replication fork, where
leading and lagging strand DNA synthesis is carried out by T7 DNA
polymerase, the 63-kDa gene 4 helicase/primase, and the gene 2.5 ssDNA-binding protein.
M13mp18 ssDNA
was purified as described (29). All nucleotides were purchased from
Amersham Corp. Synthetic dinucleotides (AC, AA, CC, CA, and dAC) and
the trinucleotide ACC were purchased from Sigma. Oligonucleotide
templates for the assay of ribo-oligonucleotide synthesis were
chemically synthesized by C. Dahl (Harvard Medical School). The
nucleotide sequences of the oligonucleotide templates are as
follows: T7-01,
5-CCTTCTCAAC TCAGGGTCAC ATATGACA-3
; T7-02, 5
-CCTTCTCAAC TCAGGGTTAC ATATGACA-3
; T7-03,
5
-CAGTGAATTC GATGACCAAG AGATACAGTC GTTCGACAGA TGACATCCAG-3
; T7-04, 5
-TTCTAGGACT ATCGGGCGAT CCCACAGTAG TAA-3
;
T7-05,
5
-CCTTCTCGCT GTGCCTTGTT TGCAGGTGCT TTAAAGATAC CACCA-3
; MR-01,
5
-TGGCGATTCG CAGTTTATAC CGATTCAGGT ACGTTAGGTA TCCATTGGTC TCCTAGGCTT AACGCCACGG-3
; MR-04,
5
-GGGTTCCAAG ACCTTAGGAT CCAGAGCCCA GCATTTGGAC TTATGGGGGG GGGGGGGGGG GTCTCCGAAG CTTGGGGGG; TK-01,
5
-TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTATCGC TAACGTTACC TAGGTAGTAA GCTTAACCTC T-3
; TK-02, 5
-ATCGCTAACG TTACCTAGGT AGTAAGCTTA ACCTCTACGT-3
; and TK-03,
5
-ACCTAGGTAA CGTTAGCGAT TACGGGATCC TCTCCGACCC GTTGGCAACC CACGTAGAGG TTAAGCTTAC T-3
. The T7 63-kDa gene 4 protein was purified as described (30). The
T7 56-kDa gene 4 protein was purified by B. Beauchamp (Harvard Medical
School) as described (18). T7 gene 5 protein-E. coli thioredoxin (1:1 complex) was purified and kindly provided by S. Tabor
(Harvard Medical School) as described (31). We refer to the 1:1 complex
of the gene 5 protein with thioredoxin as T7 DNA polymerase unless
otherwise indicated. The T7 gene 2.5 ssDNA-binding protein was purified
by D. Kong as described (32). T4 DNA polymerase, T4 polynucleotide
kinase, T4 DNA ligase, and other enzymes were purchased from Amersham
Corp.
The ability of
tetranucleotides to stimulate T7 DNA polymerase in the presence of T7
primase was measured using an M13 ssDNA template. The reaction mixtures
(20 µl) contained 40 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 µg/ml bovine serum albumin, 50 mM potassium glutamate,
0.3 mM dATP, 0.3 mM dCTP, 0.3 mM
dGTP, 0.3 mM [-32P]dTTP, 10 nM
T7 primase (hexamer), 20 nM T7 or T4 DNA polymerase, 10 nM M13 ssDNA, and the indicated amount of pACCA or pCACA
tetranucleotides. After incubation at 37 °C for 10 min, the
reactions were stopped by the addition of 2 µl of 0.2 M
EDTA (pH 8.0). The reaction mixtures were spotted onto Whatman DE81
filters, and the filters were washed four times for 10 min with 0.3 M ammonium formate (pH 8.0) followed by 98% ethanol. After
drying, the amount of [
-32P]dTMP incorporated into DNA
was measured as the radioactivity remaining on the filters by
scintillation spectrometry.
Oligonucleotide
synthesis assays using synthetic oligonucleotide templates or M13 ssDNA
were performed as described (18). The standard reactions (10 µl)
contained 40 mM Tris-Cl (pH 7.5), 10 mM
MgCl2, 10 mM dithiothreitol, 50 mg/ml bovine
serum albumin, 50 mM potassium glutamate, 0.5 mM dTTP, 0.1 mM [-32P]CTP, 10 nM T7 primase (hexamer), 10 nM M13 ssDNA or 100 nM oligonucleotides, and either 0.1 mM ATP or
0.1 mM di- or trinucleotide. After incubation at 37 °C
for 60 min, the reactions were stopped by the addition of 20 µl of
sequencing dye (98% formamide, 10 mM EDTA (pH 8.0), 0.1%
xylene cyanol FF, and 0.1% bromphenol blue). The reaction mixtures
were then heated at 95 °C for 5 min, and the labeled products were
separated by electrophoresis through 25% polyacrylamide gels
containing 8.3 M urea.
Formation of a minimal replication fork was performed as
described (11). The minimal replication fork consists of a 71-nt TK-03
circular DNA to which a 71-nt TK-01 linear DNA molecule has been
annealed through a 36-nt region complementary to TK-03. The nucleotide
sequences of the TK-01 and TK-03 oligonucleotides used for construction
of the replication fork are presented above. DNA synthesis reactions
(20 µl) using the minimal replication fork contained 40 mM Tris-Cl (pH 7.5), 10 mM MgCl2,
10 mM dithiothreitol, 50 µg/ml bovine serum albumin, 50 mM potassium glutamate, 0.7 mM dTTP, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP,
0.1 mM [-32P]CTP, 50 nM T7 DNA
polymerase, 500 nM gene 2.5 protein, and 5 nM
T7 primase (hexamer). Either 0.1 mM ATP or ApC
ribodinucleotide was present to measure de novo primer
synthesis or the extension of ApC to primers, respectively. The
reactions were started by the addition of 50 nM replication
fork and incubated at 37 °C for 5 min. The reaction was stopped by
the addition of 5 µl of 0.2 M EDTA. 5 µl of the
reaction was mixed with 15 µl of sequencing dye and applied directly
to a 25% urea gel to measure ribotetranucleotide synthesis. The
remainder of the reaction (20 µl) was deproteinized, and the DNA was
digested with BamHI and loaded onto 4% urea gels. After
electrophoresis, the DNA fragments bearing ribonucleotide primers were
visualized by autoradiography.
As discussed in the Introduction, earlier
studies had indicated that ribotetranucleotides could stimulate DNA
synthesis on ssDNA templates catalyzed by T7 DNA polymerase, provided
that the T7 primase was present (15, 25, 28). In these preliminary studies, the concentration of tetranucleotides, 100-1000
µM, far exceeded the concentration of NTPs normally used
in the primase reaction. As a result, it was difficult to evaluate the
effect of nucleotide sequence on the efficiency with which the
oligonucleotides were extended. In the experiment presented in Table
I, we examined the ability of two ribotetranucleotides,
pACCA and pCACA, to prime DNA synthesis catalyzed by either phage T7 or
T4 DNA polymerase on M13 ssDNA; ATP and CTP, the precursors for primer
synthesis, were omitted from the reaction. The tetranucleotide pACCA is
similar to the pppACCA primers synthesized by T7 primase at the
recognition site 5-TGGTC-3
; five such sites are present on M13mp18
ssDNA (33). The tetranucleotide pCACA is not synthesized by T7 primase (7, 18), although the complementary sequence 5
-TGTG-3
is present on
M13mp18 ssDNA at 12 loci. At the lowest concentration, 1 µM, pACCA was ~25-fold more efficient in promoting DNA
synthesis by T7 DNA polymerase in the presence of T7 primase than was
pCACA. At this concentration of tetranucleotide, no stimulation of DNA synthesis was detected in the absence of T7 primase. At a 10-fold higher concentration of tetranucleotide, there was a 9-fold increase in
the stimulation by pCACA as compared with the lower concentration, whereas the higher concentration of pACCA resulted in only a 1.6-fold enhancement. The less dramatic stimulation by the higher concentration of pACCA is in part due to the extent of DNA synthesis. The 298 pmol of
dTMP incorporated represents nearly complete replication of the 0.2 pmol of M13 ssDNA template (351 pmol of dTMP incorporation sites)
present in the reaction.
|
The T7 primase-dependent stimulation by the tetranucleotide is specific for T7 DNA polymerase; no significant stimulation of T4 DNA polymerase was observed. It should be noted that the use of the primer pCACA by T7 DNA polymerase is dependent on the presence of T7 primase, although this tetranucleotide is not synthesized by the enzyme in the normal priming reaction.
ADP and AMP Can Be Incorporated into the First Position of Primers Synthesized by the T7 PrimaseOligonucleotides synthesized by T7
primase contain a 5-terminal ATP since ATP is the first nucleotide
incorporated (11). In the studies presented in this report, we used
oligonucleotides lacking a 5
-terminal triphosphate. Consequently, we
examined the ability of the T7 primase to incorporate adenosine, AMP,
and ADP into the dinucleotide precursor and to extend them to tri-, tetra-, and pentanucleotides (Fig. 1).
In the presence of ATP and CTP, the T7 primase catalyzes the synthesis
of pppAC, pppACC, pppACCA, and pppACCAA on a synthetic oligonucleotide
containing the known recognition site 5-TTGGTC-3
. Both AMP and ADP
are about equally effective in supporting oligonucleotide synthesis,
but the trinucleotide cannot be extended beyond ppACC or pACC since ATP
is not available in the reaction. Although incorporation of adenosine
was greatly reduced compared with AMP and ADP, there was detectable
synthesis of AC (Fig. 1). No oligonucleotide synthesis was observed
with dAMP, dADP, or dATP.
To study the use of oligonucleotides by T7
primase uncoupled from DNA synthesis, we examined the extension of
synthetic ribodinucleotides by the enzyme. In the experiment presented
in Fig. 2A, we examined the extension of the
dinucleotide AC to ACC and ACCC on a synthetic template containing the
recognition sequence 5-GGGTC-3
by measuring the incorporation of
[
-32P]CMP. It is clear that the dinucleotide is
efficiently extended to ACC and ACCC (Fig. 2A, lane
2). The extent of synthesis is approximately the same as that
observed with ATP and [
-32P]CTP (lane 1),
and reactions containing both ATP and AC synthesize approximately equal
amounts of pppACCC and ACCC (lane 3). Therefore, the T7
primase appears to have the same affinity for ATP and AC.
Not all dinucleotides can be extended by T7 primase. As shown in Fig.
2B, the ribodinucleotide AC is extended far more efficiently than is AA, CC, or CA on either M13 ssDNA or synthetic oligonucleotide templates, each containing the complementary sequence to the
dinucleotide sequence. Each of the sequences complementary to the
extended dinucleotide is followed by a 5-guanosine residue for
labeling with [
-32P]CMP, and each contains the
3
-cryptic cytidine required by the primase for recognition:
5
-GGGTC-3
for AC, ACC, and dAC; 5
-GTTC-3
for AA; 5
-GGGC-3
for CC;
and 5
-GTGC-3
for CA. The dinucleotide AA is not extended, whereas CC
and CA are extended to CCC, CCCC, CAC, and CACC, albeit at reduced
efficiency. The trinucleotide ACC is extended to the tetranucleotide
ACCC on either template. The reaction is specific for ribodinucleotides
as evidenced by the fact that dAC is not extended on either
template.
In general, DNA primases rely upon DNA helicases
for translocation to primase recognition sites (34). The T7 63-kDa gene 4 primase has an inherent helicase activity (15, 24) and hence does not
have to interact physically with a separate helicase. The 5 to 3
translocation activity of T7 primase is dependent on the hydrolysis of
dTTP to dTDP and Pi (35, 36). As shown in Table
II, omission of dTTP from the di- or trinucleotide
extension reaction reduces the extension of AC on M13 ssDNA to <1% of
that observed in its presence. On the synthetic oligonucleotide T7-01 containing a primase recognition site, extension is reduced to ~5%
in the absence of dTTP. The greater amount of extension on the short
oligonucleotide is in keeping with earlier evidence that random
diffusion to primase sites accounts for a significant amount of primer
synthesis on short oligonucleotides (37).
|
The dinucleotide normally synthesized
in the primase reaction is AC. The preference for AC in the extension
reaction catalyzed by T7 primase suggests either that the primase can
accumulate only AC in its active site or that the reaction occurs only
at primase recognition sites on the template. If the latter supposition is correct, then extension of AC to a tri- and tetranucleotide should
occur only at template sites having the recognition sequence 5-GTC-3
,
containing the cryptic deoxycytidine.
To determine if the 5-GTC-3
site is required for dinucleotide
extension, we examined the ability of the T7 primase to extend AC and
ACC on two synthetic templates, one containing the sequence 5
-GTC-3
and the other 5
-GTT-3
(Fig. 3A). The two
templates are identical except for the substitution of T for cryptic C
in the latter template. A comparison of the extension of AC and ACC to
ACC and ACCC on the two templates shows unequivocally that the cryptic
cytidine is required. Essentially no extension of AC and ACC is
observed on the template in which the cryptic cytidine is replaced by
thymidine. Some trinucleotide extension can be observed on the latter
template, but it is only 3% of that found with the template containing
the 5
-GTC-3
site. The extension of AC and ACC on the template
containing the primase recognition sequence is even more efficient than
the synthesis of the tri- and tetranucleotides from ATP and CTP.
As shown in Fig. 2B (lanes 3 and 9), a
reduced but significant amount of tri- and tetranucleotides was
synthesized on the M13 ssDNA and on synthetic oligonucleotide templates
in the presence of the dinucleotide CC. Therefore, we also examined the
requirement of the 3-cryptic cytidine for CC dinucleotide extension.
Three templates containing the complementary sequence to CC,
5
-GGGC-3
, 5
-GGGTC-3
, and 5
-GGGTT-3
, were used in the dinucleotide
extension assay. As shown in Fig. 3B, the 5
-GGGTT-3
site
cannot support the extension of CC, whereas the 5
-GGGC-3
site does so
fairly efficiently. Inserting a thymidine residue between the cryptic cytidine and guanosine diminishes significantly the ability of the T7
primase to extend the dinucleotide. Nonetheless, the fact that the
5
-GGGTC-3
site does support some extension suggests that the enzyme
may recognize the 5
-GTC-3
site even in the absence of ATP.
For confirmation of the requirement of a primase recognition site
containing a cryptic cytidine for the extension of AC by T7 primase, we
identified the sites on M13 ssDNA at which extension occurs. The
procedure we used is based on the fact that extension of the
dinucleotide to a tetra- or pentanucleotide is required for its
function as a primer for T7 DNA polymerase (7). The primers were
labeled with [-32P]CTP in the extension reaction, and
the Okazaki fragments that were generated by T7 DNA polymerase were
digested with EcoRI, which cut M13 DNA at one site. The
resulting fragments, labeled with 32P at their 5
-termini,
were of unique lengths depending on the site at which the primer
initiated DNA synthesis. A knowledge of the M13 DNA sequence allows the
identification of the sequence at which dinucleotide extension occurred
(33).
In the presence of ATP and [-32P]CTP, the precursors
for primer synthesis, T7 primase and T7 DNA polymerase mediate the
synthesis of Okazaki fragments that, after cleavage with
EcoRI and denaturation, give rise to the bands shown in Fig.
4 (lane 1). Five major DNA fragments are
observed whose lengths (919, 1123, 1469, 2073, and 2217 nt) indicate
that they arose at positions 7150, 105, 451, 1055, and 1199 on the M13
circular DNA template. The 919- and 2217-nt fragments arise from the
two 5
-GGGTC-3
recognition sites found on M13 DNA, and the 1123-, 1469-, and 2073-nt fragments from 5
-TGGTC-3
sites. There are a number
of additional fragments that either arise from other minor recognition
sites on the M13 DNA molecule (7) or result from fragments derived from
molecules in which multiple priming events occurred. The latter give
rise to fragments whose 3
-termini is not derived from an
EcoRI cut, but rather from termination of DNA synthesis at
an adjacent Okazaki fragment.
When the coupled DNA synthesis reaction is carried out with AC in place
of ATP (Fig. 4, lane 2), both the 919- and 2217-nt fragments
are still observed, but not the 1123- and 1469-nt fragments. The
recognition site 5-TGGTC-3
found at these two latter positions cannot
give rise to a functional primer since only the trinucleotide ACC can
be synthesized from the dinucleotide in the absence of ATP, which is
needed to complete the tetranucleotide pppACCA. As was observed with
the synthetic oligonucleotide template discussed above, no fragments
are observed that would have arisen from 5
-GGGTT-3
sites or
5
-GGGTA-3
as evidenced by the absence of fragments 404, 759, and 612 nt in length.
The amount of 919- and 2217-nt fragments found in the reaction using AC is considerably less than that observed with the primase reaction using ATP and CTP. This result could reflect a requirement for a terminal triphosphate on the primer for efficient extension by T7 DNA polymerase.
Role of the T7 Primase Zinc Motif in the Extension of DinucleotidesThe T7 gene 4 protein also exists in
vivo as a 56-kDa species that has full helicase activity, but
lacks template-dependent primase activity (15, 16). The
56-kDa helicase does, however, contain the active site for
phosphodiester bond formation in that it can synthesize random
dinucleotides at low efficiency (15). As shown in Fig.
5A, the 56-kDa helicase can catalyze the
extension of AC and ACC on an M13 ssDNA template, but the amount of the extension is reduced greatly, 29- and 2.9-fold, respectively, compared
with the extension by the 63-kDa gene 4 protein. We also examined the
ability of an altered 63-kDa gene 4 protein, in which serine has been
substituted for cysteine 36 in the zinc motif, to catalyze the
extension of oligonucleotides. This altered 63-kDa protein lacks the
ability to synthesize oligonucleotides in a sequence-dependent manner, and it is unable to support T7
growth (13). As shown in Fig. 5A, the C36S mutant gene 4 protein is unable to extend dinucleotides.
Since dinucleotide synthesis catalyzed by the 56-kDa gene 4 protein is
not template-dependent (15), the cryptic cytidine residue
found in the core recognition site should not play a role in extension
by the 56-kDa gene 4 protein. The synthetic oligonucleotides T7-01 and
T7-02, containing the sequences 5-GGGTC-3
and 5
-GGGTT-3
, respectively, were used as templates. As shown in Fig. 5B,
whereas the 63-kDa gene 4 protein could efficiently extend both the di- and trinucleotides on the template T7-01, containing the cryptic cytidine, the 56-kDa gene 4 protein was able to extend only the trinucleotide. In the absence of the cryptic cytidine (template T7-02),
neither the 63-kDa nor the 56-kDa gene 4 protein could extend the
dinucleotide, but there was significant extension of the trinucleotide.
Hence, the zinc motif plays a far more significant role in the
extension of dinucleotide than in the extension of trinucleotide.
In the experiments described above, we examined the dinucleotide-dependent priming of DNA synthesis by T7 primase on ssDNA templates. Replication of the duplex T7 chromosome involves simultaneous DNA synthesis on both the leading and lagging strands at a replication fork. The economy of T7 replication is such that four proteins suffice to mediate the multiple reactions required for this process (38). T7 DNA polymerase (gene 5 protein complexed to E. coli thioredoxin) accounts for the processive polymerization of nucleotides on both strands, and T7 primase, the 63-kDa protein, provides helicase activity as well as primase activity. The fourth protein, the gene 2.5 protein, is a ssDNA-binding protein that interacts with both the DNA polymerase and the gene 4 protein and enhances their activity (39, 40).
To examine the use of dinucleotides under conditions that mimic
replication in vivo, we used a preformed DNA replication
fork, where leading and lagging strand syntheses occur simultaneously. The preformed replication fork depicted in Fig.
6A consists of a 71-nt circular ssDNA to
which a 71-nt linear ssDNA has been annealed through a 36-nt
complementary region. The resulting molecule is a partially duplex
circle bearing a 5-single-stranded tail of 35 nt. The sequence for a
BamHI site is located on the ssDNA circle, and DNA synthesis
through this sequence generates a functional BamHI
restriction site (Fig. 6A). In addition, the ssDNA circle contains two sequences (5
-AACCC-3
and 5
-GACCC-3
) that, when copied
by DNA polymerase, generate the sequences 5
-GGGTT-3
and 5
-GGGTC-3
.
The latter sequence is the primase recognition sequence, whereas the
former lacks the cryptic cytidine residue required for recognition. If
DNA synthesis is initiated from tetranucleotide primers generated at
the 5
-GGGTC-3
site, cleavage with BamHI will generate a
60-nt fragment. Any dinucleotide extended to a functional primer at the
5
-GGGTT-3
sequence will give rise to a 49-nt fragment (Fig.
6A).
In the experiment shown in Fig. 6B, the circular molecule
was replicated using T7 DNA polymerase, T7 primase, and the T7 gene 2.5 protein in the presence of [-32P]CTP and either ATP or
AC. After incubation for 10 min, the product was isolated and digested
with BamHI, and the resulting fragments were resolved on a
4% gel. In the reactions containing either ATP or AC, only the 60-nt
fragment was observed, indicating that dinucleotides were extended to
functional tetranucleotide primers at the primase recognition sequence
containing the 3
-cryptic cytidine (Fig. 6B).
We also examined the relative efficiency with which ATP and AC are used
in the T7 primase reaction in the presence of
[-32P]CTP (Fig. 6C). When ATP, AC, and CTP
are added together to a reaction mixture containing the replication
fork, ACC, ACCC, pppACC, and pppACCC are synthesized at comparable
rates. Thus, it appears that the T7 primase has very similar affinities
for ATP and AC.
Early studies of the gene 4 proteins encoded by bacteriophage T7
showed that they stimulated the use of ribotetranucleotides as primers
by T7 DNA polymerase (15, 25). In this study, we extend these findings
to show that T7 DNA polymerase uses ribotetranucleotides as primers far
more efficiently in the presence of the T7 primase than in its absence.
Furthermore, ribodinucleotides can be extended by the primase in the
presence of the appropriate nucleoside triphosphate, CTP, to yield
functional tetranucleotide primers. Remarkably, the use of
dinucleotides is dictated by their sequence; the dinucleotide 5-AC-3
,
the dinucleotide synthesized by the T7 primase at its basic recognition
site, 5
-GTC-3
, is strongly preferred. Our data further show that the
extension of the dinucleotide to a tetranucleotide occurs only at a
primase recognition site such as 5
-GGGTC-3
. Thus, the cryptic but
essential cytidine residue that composes a portion of the recognition
sequence is essential; no extension of AC occurs on templates
containing the sequence 5
-GGGTT-3
. Evidently, the same requirements
that dictate primer synthesis from ATP and CTP precursors pertain to
the extension of synthetic oligonucleotides by the T7 primase.
The precise mechanism by which the dinucleotide AC is selected by the T7 primase and extended at a primase recognition site is not known. It is clear from our studies that the Cys4 zinc motif of the T7 primase is essential for the extension of the AC dinucleotide in that neither the 56-kDa gene 4 helicase, which lacks this domain, nor a genetically altered T7 primase in which one of the cysteines of the zinc motif has been replaced by serine can catalyze the extension. However, these studies do not allow us to distinguish between two possibilities regarding the mechanism by which the dinucleotide arrives at the primase recognition site. In one scheme, the AC dinucleotide transiently binds to all GT sequences on the template, but is stably bound only when the GT sequence lies within a primase recognition site that is already occupied by a primase molecule. In the second scenario, the T7 primase binds the dinucleotide; the complex then translocates until it reaches a primase recognition site, where it is extended. In both instances, the translocation of the primase along ssDNA plays a crucial role; in fact, we find that omission of dTTP, the nucleotide required for translocation, drastically reduces extension of the dinucleotide.
Of the two possibilities mentioned above, we favor the second, in which
the dinucleotide is first bound to the enzyme and transported to the
site. The competition experiments described here have shown that the AC
dinucleotides are used for tetranucleotide synthesis equally as well as
ATP when present together at identical concentrations. Furthermore, it
seems unlikely that, at the low concentrations of dinucleotides used in
these experiments, annealing to the template would occur to an extent
that the rapidly translocating primase (300 nt/s) would ever encounter
one bound at a recognition site. Finally, cross-linking studies by
Mustaev and Godson (41) using the DNA primase of E. coli,
the DnaG protein, have shown that ATP, the 5-terminal nucleotide of
primers synthesized by the enzyme, binds to the enzyme prior to binding
to ssDNA. If this result is also applicable to the T7 DNA primase, then
the T7 primase would arrive at a recognition site with an ATP already positioned in its active site.
The 3-cryptic cytidine of the 5
-GTC-3
recognition site clearly plays
an important role in the specific extension of AC to the trinucleotide
ACC, as is the case when ATP is the initiating nucleotide. Although the
significance of the 3
-cytidine residue in oligonucleotide extension
depends on the length of the oligonucleotide to be extended, it is
nonetheless very important in the extension of the trinucleotide ACC.
For example, in the experiment shown in Fig. 3, the ability of the
primase to extend ACC at the sequence 5
-GGGTT-3
, lacking the cryptic
cytidine residue, was only 3-fold greater than its ability to extend
AC. Cross-linking studies have clearly shown that both the DnaG primase
and the
-subunits of E. coli RNA polymerase maintain
contact with the first nucleotide of the newly synthesized RNA during
the polymerization of the first several nucleotides (41, 42). Thus, the
association of the primase with the cryptic cytidine may increase the
stability of the complex; the dependence on this association to
stabilize the complex, however, may be reduced slightly as the length
of the primer increases.
An unexpected finding is that both ADP and AMP could replace ATP as the
first nucleotide incorporated by the T7 primase. This observation is
not too surprising in light of cross-linking studies with E. coli DnaG primase in which the triphosphate of the first nucleotide was chemically modified and the modified ATP was
incorporated by the enzyme (41). However, although the pAC and AC
dinucleotides can be readily extended to the tetranucleotide, it is not
yet clear if the absence of a triphosphate affects the subsequent extension of the primer by the DNA polymerase. In the experiments presented in Figs. 4A and 6B, the AC dinucleotide
appeared less efficient than ATP in supporting lagging strand DNA
synthesis. Whether or not triphosphates alone or a mixture of mono-,
di-, and triphosphates are found at the 5-termini of Okazaki fragments synthesized in vivo is at present not known since the
in vivo experiments on primer synthesis in T7-infected cells
did not address this point (6). Our in vitro results suggest
that the presence of these various groups would depend on the
intracellular pool of AMP, ADP, and ATP. In any case, the 5
-terminal
nucleotide along with the remainder of the RNA primase must be removed
prior to ligation. Both the T7 gene 6 exonuclease and the 5
to 3
exonuclease activity of E. coli DNA polymerase I have no
difficulty in removing such terminal RNA with a 5
-triphosphate
(43).
The large amounts of dinucleotides relative to the functional
tetranucleotide primers that arise during primer synthesis catalyzed by
the T7 primase both on synthetic oligonucleotides and on M13 ssDNA
remain puzzling. Clearly, some arise as intermediates in the synthesis
of the tetranucleotide at primase recognition sites as shown by their
appearance on synthetic templates that have only a complete primase
recognition site (18). On templates such as M13 DNA, however, the
dinucleotides can arise from the many 5-GTC-3
sequences that
represent the basic primase recognition site, but yet do not have the
proper 5
-sequence that can support extension of the dinucleotide. Why
dinucleotides are synthesized at sites where they cannot be extended to
functional primers is not known. It is possible that mechanisms exist
in vivo to curtail their synthesis at such sites. Another
possibility is that the dinucleotides synthesized at the basic
recognition sites remain bound to the primase, as we suggested above,
and are thus properly positioned when the primase reaches a complete
primase recognition site, where they can be extended to a
tetranucleotide.
It is also possible that, during replication of the leading and lagging strands at the replication fork, the action of the helicase/primase hexamer is modulated by its interaction with T7 DNA polymerase and the T7 gene 2.5 ssDNA-binding protein. In this study, we used a preformed replication fork containing a single primase recognition site. In the presence of T7 DNA polymerase, the 63-kDa gene 4 helicase/primase, and the T7 gene 2.5 ssDNA-binding protein, both leading and lagging strand syntheses occur simultaneously. Under these conditions, in the presence of ATP and CTP, the single primase recognition site is used by T7 DNA primase, as previously shown to be the case on ssDNA (18). Although unextended dinucleotides are observed to accumulate during the replication of this molecule, the ratio of tetranucleotides to dinucleotides is increased over that normally seen on ssDNA, suggesting that the utilization of dinucleotides by the primase is more efficient at a replication fork.
We are grateful to Benjamin B. Beauchamp, Joonsoo Lee, Daochun Kong, and Stanley Tabor for providing purified proteins and helpful discussions. We are also very grateful to Ingrid Richardson and Khandan Baradaran for comments on and constructive criticisms of the manuscript.