(Received for publication, October 11, 1996, and in revised form, December 10, 1996)
From the Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115
The 63-kDa gene 4 DNA primase of phage T7
catalyzes the synthesis of oligoribonucleotides on single-stranded DNA
templates. At the sequence, 5-GTC-3
, the primase synthesizes the
dinucleotide pppAC; the cytidine residue of the recognition sequence is
cryptic. Only tetraribonucleotides function as primers, but the
specificity for the third and fourth position is not as stringent with
a preference of CMP > AMP
UMP > GMP. The predominant
recognition sites on M13 DNA are 5
-(G/T)GGTC-3
and 5
-GTGTC-3
.
Synthesis is usually limited to tetranucleotides, but T7 primase can
synthesize longer oligoribonucleotides on templates containing long
stretches of guanosine residues 5
to the recognition sequence. The
specificity beyond the first two positions of the primer increases as
the length of the template on the 3
-side of 5
-GTC-3
increases. On an
oligonucleotide having 20 3
-flanking cytidine residues GMP is
incorporated at the third position; incorporation is reduced 4-fold
when the flanking sequence reaches 65 residues, and little is
incorporated on M13 templates. The presence of the 56-kDa gene 4 helicase decreases the incorporation of GMP on long templates. We
propose that pausing is required for the incorporation of less preferred nucleotides and that pausing is decreased by the ability of
the primase to translocate 5
to 3
on templates having long 3
-flanking sequences.
Gene 4 of bacteriophage T7 encodes two co-linear proteins, a 56- and a 63-kDa protein (1). In phage-infected cells, the two proteins are
expressed in approximately equal amounts from two in-frame translation
initiation sites 189 bases apart on the gene 4 transcript. Thus, the
56-kDa gene 4 protein lacks the 63 N-terminal amino acid residues of
the 63-kDa gene 4 protein. The 56-kDa protein (helicase) binds to
single-stranded DNA (ssDNA)1 in the
presence of a nucleoside triphosphate (dNTP) and then, in a reaction
coupled to the hydrolysis of the dNTP, translocates 5 to 3
along the
ssDNA (2, 3). Upon encountering a duplex region of DNA, its continued
translocation unwinds the duplex (2).
The 63-amino acid N-terminal domain of the 63-kDa gene 4 protein (primase) contains a Cys4 zinc-binding motif (4, 5), a domain that enables the primase to synthesize oligoribonucleotides in a template mediated reaction. Although the zinc-binding motif is essential for primer synthesis (5), it alone cannot account for the ability of the protein to recognize specific sequences on ssDNA (6). Gene 4 protein is essential for phage growth and physically interacts with both the gene 5 DNA polymerase and the gene 2.5 ssDNA binding protein to mediate leading and lagging strand synthesis at the T7 replication fork (7-12). The 63-kDa gene 4 protein has helicase activity as well as primase activity, and it alone can support the growth of T7 bacteriophage in vivo (13, 14).
DNA primases are responsible for the synthesis of oligoribonucleotides
that serve as primers for DNA polymerases on the lagging strand of the
replication fork (15). The gene 4 primase of phage T7 catalyzes the
template- directed synthesis of di-, tri-, tetra-, and
pentaribonucleotides on ssDNA (16-19). However, both in
vivo and in vitro only tetra- and pentaribonucleotides
can serve as functional primers for T7 DNA polymerase (17, 20). Studies employing synthetic oligonucleotide templates have demonstrated that
the trinucleotide sequence 5-GTC-3
is both necessary and sufficient
for the protein-DNA interaction required to initiate oligoribonucleotide synthesis (19). The 3
-cytidine is required for
recognition but is not copied into the primer. Although limited synthesis of pppAA dinucleotide has been observed to occur at the
trinucleotide sequence 5
-TTC-3
under certain conditions, the pppAA
dinucleotide is not extended further (6, 21). Analysis of the
oligoribonucleotide primers attached to the 5
-end of Okazaki fragments
both in vivo and in vitro has shown that the T7
primase synthesizes predominantly pppAC(C/A)(C/A) tetraribonucleotides; UMP and GMP are not found in the primers (17, 18, 20).
Gene 61 primase of bacteriophage T4 and DnaG primase of
Escherichia coli also contain a single Cys4 type
zinc motif at their N termini (5, 22). Both the T4 and E. coli primases recognize trinucleotide sequences, 5-G(T/C)T-3
and
5
-CTG-3
, respectively (23-27), and the 3
-nucleotide of the sequence
is not copied into the primer. In contrast to these similarities among
the three primases, the T4 and E. coli primases differ from
the T7 primase in that the template sequence 5
to the trinucleotide
recognition sequence is less stringent both in regard to sequence and
to the length that is copied (23, 25, 26, 28). As a result the oligoribonucleotides found at the 5
-termini of Okazaki fragments synthesized by the T4 and E. coli primases are
ppp(A/G)C(N)3 or pppAG(N)9-11, respectively
(28-30). In this report we examine in detail the specificity of
nucleotide selection by the T7 primase.
The T4 and E. coli primase systems also differ from that of
T7 in one other striking manner. Whereas the T7 63-kDa gene 4 protein
catalyzes both helicase and primase activities, the T4 and E. coli helicase and primase activities reside within separate polypeptides (28, 31-34). As a result the T7 primase is faced with the
dilemma that upon reaching a primase recognition site it must either
pause in its 5 to 3
translocation and synthesize a primer in the
opposite direction or continue its role as a helicase. In this report
we present evidence that the inherent translocation activity of the T7
primase plays a role in determining which potential sites in the
template are used for oligoribonucleotide synthesis.
The T7 63-kDa gene 4 protein used in these studies has Met at position 64 replaced by Gly but this protein has all of the catalytic properties of the wild-type protein (35, 36). Wild-type T7 56-kDa gene 4 protein was purified by B. Beauchamp (Harvard Medical School) as described previously (19). T7 gene 5 DNA polymerase was purified as described previously (11) and kindly provided by S. Tabor (Harvard Medical School). T7 gene 2.5 ssDNA binding protein was purified by D. Kong (Harvard Medical School) as described previously (37). T4 polynucleotide kinase, T4 DNA ligase, other enzymes, and nucleotides were purchased from Amersham Corp.
DNA TemplatesM13mp18 ssDNA was purified as described
elsewhere (38). Oligonucleotide templates for assay of
oligoribonucleotide synthesis by T7 DNA primase were chemically
synthesized by C. Dahl. (Harvard Medical School). The nucleotide
sequences of the DNA templates are presented in Table I. The six
oligonucleotides labeled HR3-5 to HR3
-65 were prepared by annealing
5
-AAAAAAAAAAGACGC-3
, 5
-CCCCCCCCCCCCCCCGCGTCTTTTT-3
, and poly(dT) in
a ratio of 1:1:1. The poly(dT) used in separate reactions varied from 5 to 65 nucleotides in length. After ligation the products were separated
on 10% urea-gel, and the DNA fragments corresponding to 25, 30, 35, 45, 65, and 85 nucleotides (nt) in length were isolated.
|
Oligoribonucleotide synthesis catalyzed by DNA primase was determined by measuring the amount of radioactively labeled oligoribonucleotide synthesized (19). The standard reaction (10 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, 0.5 mM dTTP, 0.1 mM rNTPs, 10 nM M13 ssDNA or 100 nM oligonucleotides, and 60 nM (monomer) primase unless otherwise indicated. After incubation at 37 °C for 60 min, the reaction was stopped by the addition of 20 µl of sequencing dye (98% formamide, 10 mM EDTA (pH 8.0), 0.1% xylene cyanol FF, 0.1% bromphenol blue). The reaction mixtures were then heated at 95 °C for 5 min. The products were separated by electrophoresis through 15 or 25% gels containing 50% urea.
DNA Synthesis and Primer Synthesis at a Minimal Replication ForkFormation of a minimal replication fork was carried out as
follows. One nanomole of RF-1 oligonucleotide (71 nt) was
phosphorylated using T4 polynucleotide kinase and mixed with 1 nmol of
RF-3 (40 nt) in 100 mM Tris-Cl (pH 7.5). The mixture was
heated at 95 °C for 5 min and then slowly cooled to anneal, followed
by ligation of the oligonucleotides by incubation overnight at 16 °C
using T4 ligase. The 71-nt closed circular DNA was isolated on a 10% urea-gel and then annealed with RF-2 oligonucleotide (75 nt) at a molar
ratio of 1:3 at 65 °C followed by cooling overnight. Reactions using
the minimal replication fork (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, 0.7 mM dTTP, 0.2 mM d(A,G,C)TPs, 0.1 mM
[-32P]CTP, 0.1 mM ATP or (A,G,U)TPs, 50 nM gene 5 DNA polymerase, 500 nM gene 2.5 protein, and 5 nM (hexamer) primase. The reactions were
started by adding the replication fork to 25 nM. After
incubation at 37 °C for 5 min, the reactions were stopped by the
addition of 5 µl of 0.2 M EDTA. A portion of the reaction
mixture (5 µl) was mixed with 15 µl of sequencing dye and applied
directly to a 25% urea-gel for measurement of oligoribonucleotide
synthesis. The remainder of the reaction (20 µl) was deproteinized,
and the DNA was precipitated with ethanol, digested with
BamHI, and loaded onto a 6% urea-gel. After electrophoresis
the DNA fragments containing ribonucleotide primer were visualized by
autoradiography.
The primers used by T7 DNA polymerase both in vivo and
in vitro are exclusively tetra- or pentaribonucleotides (18,
20). However, in vitro both di- and triribonucleotides are
synthesized by the 63-kDa gene 4 primase (16, 19). We have previously shown that the minimal recognition sequence for the T7 primase is
5-GTC-3
and at this site the gene 4 protein catalyzes the synthesis
of the dinucleotide pppAC unless the proper nucleotides are present in
the template for extension to tri- and tetranucleotides (19). Analysis
of the sequence of the primers attached to the 5
-termini of Okazaki
fragments synthesized in vivo and in vitro (18,
20) as well as the characterization of the nucleotides incorporated by
the primase on synthetic oligonucleotide templates (6, 19, 21) revealed
that pppAC is the only dinucleotide that can be extended to tetra- or
pentanucleotide primers. The dinucleotides pppAA and very few pppGN are
synthesized on M13 ssDNA but the pppAA is not extended to the
trinucleotide (21). Although the nucleotides found at the first and
second position of the functional primer are invariant, the specificity
of subsequent nucleotides in the primer as well as the factors
controlling the length of the primers have not been examined
rigorously. Previous studies have demonstrated that although there is a
strong preference for CMP and AMP to be incorporated after the first
two nucleotides, the stringency for nucleotide incorporation is less at
these positions (6, 21).
In order to determine the frequency with which each nucleotide is
incorporated into primer, we have carried out oligonucleotide synthesis
in reactions containing either M13 ssDNA or synthetic oligonucleotide
templates and one of each of the four [-32P]NTPs
in the presence of the other three unlabeled rNTPs. Using the
7249-nt M13mp18 ssDNA and much shorter synthetic oligonucleotides, we
demonstrated that nucleotide specificity is influenced by the length of
the template. In addition, short oligonucleotide template enabled the
examination of nucleotide incorporation at specific sequences.
As shown in Fig.
1, A and B, when M13 ssDNA was
used as a template, incorporation of GMP and UMP by gene 4 primase
after the second position in the oligoribonucleotide was only 6.7 and
13.8%, respectively, compared with that of CMP. AMP was incorporated as well as CMP. In agreement with earlier studies pppAC and pppAA dinucleotides were synthesized, but only pppAC was extended (Fig. 1A). No significant amount of dinucleotide containing GMP or
UMP was observed. As described above GTP can be used as the first nucleotide of the primer, albeit at very low frequency. Consequently, a
portion of radioactivity observed with [-32P]GTP is
most likely due to incorporation of GTP as the first nucleotide. In
addition to pppACC and pppACA trinucleotides in the middle two lanes,
pppACU trinucleotide was also observed in the last three lanes.
However, the pppACU trinucleotides were less efficiently extended to
tetranucleotide primers. These results demonstrate that the gene 4 primase prefers CMP and AMP for incorporation after the second position
of the primer. Although UMP can be incorporated, the resulting
trinucleotides are less likely to be further extended.
Although the length of the majority of primer synthesized by the gene 4 protein are limited to five nucleotides, further extension can occur as
evidenced by the presence of a small amount of hexamer and heptamer
seen in Fig. 1A. Since the gene 4 primase incorporates GMP
and UMP poorly, this property may play a role in limiting the length of
the oligoribonucleotide synthesized by the primase starting from a
basic recognition site. In fact, inspection of the M13 ssDNA sequences
reveals that the longest stretch of guanosine or thymidine residues
following the recognition sequence 5-GTC-3
is 5
-TGGGGGTC-3
, a site
that yields the heptanucleotide pppACCCCCCA observed in Fig.
1A. In order to determine whether the T7 primase has an
inherent property limiting the length of primers synthesized to four or
five nucleotides or if the length of the primer is, at least in part,
determined by the DNA sequence, we constructed a template containing a
repeat of 19 guanosine residues on the 5
-side of the primase
recognition site. On this template, T7 primase synthesizes long
primers, up to 22 nt in length (Fig. 2). The presence of
an additional nucleotide suggests that the primase adds a one
nucleotide overhang upon reaching the 5
-end of the template strand.
The amount of oligoribonucleotide synthesized increased linearly, up to
60 min, and the proportional amount of short and long primers was
roughly constant over this period.
Ribonucleotide Specificity at Third and Subsequent Positions of Primers Synthesized on Synthetic Oligonucleotide Templates
As
shown in the previous section, gene 4 primase incorporates
predominantly CMP and AMP after the second position in primers synthesized on M13 ssDNA. In order to compare more precisely the ability of the T7 primase to incorporate each of the four nucleotides at positions beyond the initial dinucleotides we have examined primer
synthesis on synthetic oligonucleotides of known sequence. The
templates shown in Fig. 3 varies in length from 25 to 80 nt, and they all contain the basic primase recognition site 5-GTC-3
with variation in the sequence occurring on the 3
-side of the recognition sequence (see Table I for complete
sequences). Oligoribonucleotide synthesis reactions were carried out on
the various templates shown in Fig. 3 in the presence of ATP, GTP, UTP,
and [
-32P]CTP. To our surprise when these
oligonucleotides were used as templates, GMP and UMP could be readily
incorporated at the third and subsequent positions (Fig.
3A). The total amount of oligoribonucleotides having lengths
greater than two residues is presented in Fig. 3B for each
template. GMP (template 8) and UMP (template 9)
can be incorporated into the third position of the primer when a
cytidine or thymidine residue follow the basic recognition site in the template. Furthermore, these nucleotides can also be incorporated into
the fourth position (templates 5 and 7,
respectively). Incorporation of UMP is relatively low, but the amount
incorporated is still comparable to that of CMP incorporated (compare
template 1 and 9, Fig. 3, A and
B). The relatively low synthesis of oligonucleotides on
templates 1 and 2 is most likely due to length of the sequence 3
to
the recognition sequence 5
-GTC-3
on these templates (see below).
Interestingly, in the case of template 8, once two GMP residues have
been incorporated into the third and fourth positions, the
oligoribonucleotide is efficiently extended as evidenced by the
accumulation of primers greater than four in length. Thus the presence
of guanosine on the 3
-terminus of the primer does not inhibit its
further extension by the primase. Two different primase recognition
sites 5
-AGGTC-3
and 5
-ATGTC-3
are present in template 7, necessitating the incorporation of a uridine residue at the fourth
position of the primer. Tetranucleotides are synthesized with equal
efficiency on both.
We have examined the ability of the T7 primase to use templates
containing adenosine or cytidine in the fourth position of the template
sequence, 5-G(A/C)GTC-3
, in the presence of a template containing the
sequence, 5
-GGGTC-3
as a competitor. As shown in Fig.
4, all three templates alone were approximately equal in
their ability to incorporate CMP, GMP, or UMP at the third position of
the oligoribonucleotides. Interestingly, the oligoribonucleotides containing either GMP or UMP at the third position were far more efficiently extended to the 5
-end of the polyguanosine-containing template than those incorporating CMP at this position (see
"Discussion"). When each of the templates containing adenosine or
cytidine at the fourth position of the recognition sequence were mixed
with an equal amount of template containing guanosine at this position, the amount of primer synthesis was reduced by half. Since the reactions
were performed under conditions of template excess, a reduction in the
incorporation of GMP and UMP by 2-fold indicates that the adenosine or
cytidine containing templates are used as well as the guanosine
containing template.
Fidelity of T7 DNA Primase Varies According to Position in Template
The three extensively studied prokaryotic DNA primases, E. coli DnaG protein, T4 gene 61 protein, and T7 gene 4 protein, all recognize a basic trinucleotide sequence in the template (23-27). Consequently, primers synthesized by all these primases have essentially invariant nucleotides in the first two positions. Furthermore, like the T7 primase described above, both the E. coli (25, 26, 30, 39) and T4 (23, 24) primases relax this stringency after the first two nucleotides are incorporated. These properties suggest that the mechanism by which the first two nucleotides are incorporated differ from that involved in subsequent incorporation. Furthermore, formation of the first phosphodiester bond may well require that the primases bind tightly to both the first and the second nucleotide, thus dictating a specific nucleotide at each binding site. However, as extension of the dinucleotide progresses, the primase then interacts with the growing oligoribonucleotide annealed to the template and the next incoming nucleotide. The kinetics and specificity for the two steps would be expected to differ significantly. In fact, Swart and Griep (39) have shown that synthesis of dinucleotides is a rate-limiting step for total oligoribonucleotide synthesis by the E. coli primase.
If the low stringency incorporation of DNA primases at positions after
the second nucleotide is brought about by the difference in the
stability of the primase/template/nucleotide complex, it is also
possible that the fidelity of the enzyme at a given site in the primer
varies with its relative distance from the basic recognition site
5-GTC-3
. The high fidelity of T7 primase at the first and the second
position of the primers is apparent from the fact that ATP and CMP are
the only nucleotides found at the first and second positions of the
primer, respectively, on M13 ssDNA, although there are rare exceptions
as described in the Introduction. We have examined oligoribonucleotide
synthesis on five templates in which each of the guanosines between the
fourth and the eighth position of the recognition sequence
5
-GGGGGGTC-3
(indicated by boldface letters) is replaced
by an adenosine. The products of oligoribonucleotide synthesis on each
template were identified by gel electrophoresis (Fig.
5A). When the oligoribonucleotides were
labeled with [
-32P]UTP in the presence of all four
NTPs, UMP was incorporated at the position corresponding to the
adenosine in all five templates (last five lanes in Fig.
5A). When the oligoribonucleotides were labeled with
[
-32P]CTP in the presence of all four rNTPs, there was
no misincorporation of CMP at the third position as evidenced by the
absence of a band containing pppACC (lane 1), a small
misincorporation at the fourth position corresponding to pppACCC
(lane 2), and significant misincorporation at the fifth
position (pppACCCC; lane 3) following the 5
-GTC-3
recognition site. Further, when UTP and GTP were removed from the
reaction, CMP was again not misincorporated at the third position
(lane 6), but was readily incorporated after the third
position (lanes 7-10). Thus at the third position of the
primer there is no misincorporation of CMP opposite adenosine (lanes 1 and 6) or UMP opposite guanosine
(lane 12), whereas at the fourth position CMP opposite
adenosine (lanes 2 and 7) is misincorporated, and
UMP opposite guanosine (lane 13) is not. After the fourth
position misincorporation of CMP is frequent, whereas UMP is not.
In order to confirm that T7 gene 4 primase loses fidelity in proportion
to its distance from the basic recognition site 5-GTC-3
, we examined
the frequency of misincorporation of CMP at a position nine residues
from the recognition site. On the template
5
-G10NG8GTCN10-3
in which each of
the four nucleotides are located at the N9 position, CMP was
misincorporated opposite adenosine (Fig. 5B, lane
2), thymidine (lane 3), and cytidine (lane
4). Compared to the correct incorporation of CMP opposite to
guanosine (lane 1), CMP was misincorporated opposite
adenosine, thymidine, and cytidine (lanes 2-4)
approximately 50, 30, and 80%, respectively. In the absence of UTP and
GTP, CMP was misincorporated more frequently opposite thymidine, and cytidine (last four lanes in Fig. 5B). Both in the absence
and presence of UTP and GTP, the misincorporation of CMP opposite thymidine and cytidine (lanes 3 and 4,
lanes 7 and 8) resulted in less extension beyond
the misincorporated nucleotide.
The results
presented above (Figs. 1 and 3) show that the T7 primase relaxes its
stringency on oligonucleotide templates as compared to an M13 template.
For example, oligonucleotides containing the recognition sequence
5-CCGTC-3
are efficiently used as template for tetranucleotide
synthesis whereas this sequence is seldom used on M13 ssDNA. One
difference between the two types of templates is the greater length of
DNA surrounding the basic trinucleotide recognition site 5
-GTC-3
in
M13 ssDNA. In the case of the 7249-nt M13mp18 ssDNA molecule, the gene
4 protein is likely to search for recognition sites by using its
inherent helicase activity to track along the DNA 5
to 3
(35),
whereas in the case of oligonucleotide templates, random diffusion to
the sites may play a more important role. Consequently, at those sites
where CTP or ATP would not normally be used, the rapid movement of the
protein through these sites by translocation would not allow sufficient time for incorporation of a nucleotide such as GMP.
In order to examine the effect of flanking sequences on nucleotide
usage, we designed six templates each having a different length of DNA
on the 3-side of the potential recognition site 5
-GCGTC-3
(Fig.
6A). Oligoribonucleotide synthesis was then measured in reactions containing the 63-kDa gene 4 protein that has
both primase and helicase activity and in reactions containing equal
amounts of the 56- and 63-kDa gene 4 protein. The 56-kDa gene 4 protein
has only helicase activity. As shown in Fig. 6, B and
C, the synthesis of oligoribonucleotides containing GMP increased as the length of the 3
-flanking sequence approached 15 nucleotides. Most likely this effect of length on primer synthesis reflects an optimal size for binding to the recognition site since there was a similar effect on dinucleotide synthesis. Flanking sequences greater than 15, however, resulted in a decrease in the
synthesis of oligoribonucleotides containing GMP, the effect being
proportionate to length. In contrast, dinucleotide synthesis is not
affected to the same extent. This effect is accentuated by the presence
of additional helicase augmented by the presence of 56-kDa gene 4 protein.
Use of Primers Containing a Guanosine Residue at a Replication Fork
Tetranucleotides synthesized at the sites 5-(G/T)GGTC-3
and 5
-GTGTC-3
are known to serve as primers for lagging strand synthesis both in vivo (20) and in vitro (17,
18). In as much as the T7 primase can also synthesize
oligoribonucleotides containing a guanosine residue at recognition
sites such as 5
-TCGTC-3
, it was of interest to determine if these
sites are used during simultaneous synthesis of leading and lagging
strands at a replication fork and, if so, whether they are used as
primers by T7 DNA polymerase. To address this question we have used a
preformed replication fork consisting of a single-stranded circular DNA
molecule of 71 nucleotides to which a 71-nt oligonucleotide whose
3
-36-nt terminus is complementary to the circular DNA has been
annealed. The resulting preformed replication fork depicted in Fig.
7A consists of a partially duplex circle with
a 5
-ssDNA tail of 35 nt. This 5
-single stranded terminus is required
for the loading of the gene 4 protein (40). The preformed replication
fork also contains two gene 4 protein recognition sites, 5
-GGGTC-3
and 5
-TCGTC-3
that would give rise to primer synthesis on the linear,
lagging strand. On such a preformed replication fork, leading strand
synthesis can be achieved by T7 DNA polymerase, T7 gene 4 protein, and
the four dNTPs (40, 41). Upon addition of the four NTPs, primer synthesis by the 63-kDa gene 4 protein and their extension by T7 DNA
polymerase should result in lagging strand synthesis. T7 gene 2.5 protein stimulates lagging strand synthesis (10).
As shown in Fig. 7B, under conditions that give rise to
leading and lagging strand synthesis oligoribonucleotides are
synthesized by the gene 4 primase as measured by the incorporation of
[-32P]CMP into di-, tri-, and tetranucleotides.
However, the only products were pppAC, pppACC, and pppACCC.
Since the pppACC was efficiently extended to pppACCC under these
conditions, less pppACC accumulation was observed in Fig.
7B. No oligonucleotide-containing guanosine, such as pppACG
or pppACGA was observed, suggesting that the 5
-TCGTC-3
site was not
used for primer synthesis during simultaneous DNA synthesis.
However, in this experiment a significant portion of the
oligoribonucleotides synthesized were used as primers by T7 DNA
polymerase on the lagging strand. As a result, any primers extended
would not be observed in Fig. 7B. In order to determine if
any oligoribonucleotides containing GMP were used as primers, the
product DNA-containing primers labeled with [-32P]CMP
were digested with BamHI. With BamHI digestion,
Okazaki fragments synthesized from the 5
-GGGTC-3
or 5
-TCGTC-3
site resulted in a 60- or 49-nt fragment, respectively (Fig. 7A).
As shown in Fig. 7C, in addition to the 60-nt fragment, a
small percentage of the 49-nt fragment whose 5
-oligoribonucleotide
contains GMP at the third position of the primer was found. Therefore,
the 5
-TCGTC-3
site is used at a low frequency, and most of the
primers arising from the 5
-TCGTC-3
site are extended by T7 DNA
polymerase during synthesis of leading and lagging strands at the
replication fork.
The DNA primase of bacteriophage T7, the product of the viral gene
4, catalyzes the synthesis of oligoribonucleotides for use as primers
by T7 DNA polymerase (16, 17). Although the sequence of the
oligoribonucleotides is dictated by that of the single-stranded
template (18), neither the initiation of primer synthesis nor the
length of the primer is random. In fact, the majority of the primers
found at the 5-termini of Okazaki fragments synthesized in
vivo are tetra- or pentanucleotides of three predominant sequences. In this report we address some of the parameters that dictate the sites of primer synthesis.
In confirmation of earlier studies (19) we find that T7 gene 4 primase
is stringent in its requirement for a trinucleotide recognition site,
5-GTC-3
. On either single-stranded M13 DNA or synthetic
oligonucleotides, all oligoribonucleotides synthesized by the T7 gene 4 primase contain pppAC at their 5
-termini (16-19); the 3
-terminal
cytidine is cryptic in that it is not copied into the primer.
Furthermore, the trinucleotide sequence 5
-GTC-3
is sufficient for
recognition by the T7 primase, and templates containing this sequence
support the synthesis of the dinucleotide pppAC (19). The dinucleotide
synthesized at the trinucleotide recognition site, however, are not
used by T7 DNA polymerase as primers but instead must first be extended
to tetra- or pentanucleotides (17, 18, 20).
The precise mechanism by which the T7 primase and other prokaryotic primases recognize a trinucleotide sequence on ssDNA is at present unknown. A major determinant is undoubtedly the Cys4 type zinc motif found in all DNA primases (5, 22). In the case of phage T7 a single amino acid substitution of Ser for each of the four conserved Cys residues renders the primase inactive (5). The zinc motif is not the sole determinant for recognition, however, since chimeric DNA primases in which the zinc motif of the T7 primase has been replaced with those from the phage T4 and E. coli primases do not recognize the trinucleotide recognition site of any of the three primases, although they do still recognize a trinucleotide sequence (6).
Since the average length of Okazaki fragments (lagging strand DNA
fragments synthesized from gene 4 primers) is 1000-2000 nucleotides,
it is clear that parameters other than the basic trinucleotide
recognition site affect the location of primer synthesis (10,
41).2 One important determinant is the
identity of the nucleotides 5 to the trinucleotide recognition site.
For example, both in vivo (20) and in vitro (18)
studies show that the majority of primers are synthesized from three
sites, 5
-GGGTC-3
, 5
-TGGTC-3
, and 5
-GTGTC-3
. In these studies a
number of other minor sites were observed. These minor alternative
sites always contained the basic trinucleotide recognition sequence at
their 3
-termini, departing from the three predominant sites only with
regard to the two nucleotides on the 5
-side of the trinucleotide
recognition site. Furthermore, the percentage of oligoribonucleotides
that actually function as primers for Okazaki fragment synthesis
in vitro is quite low even in the presence of excess amounts
of T7 DNA polymerase.3
An interesting observation that arose during the determination of
nucleotide specificity was the ability of the T7 primase to synthesize
oligoribonucleotides efficiently on synthetic oligonucleotides containing the sequence 5-NNGTC-3
, while on M13 DNA there is a clear
preference for the recognition sites 5
-GGGTC-3
, 5
-TGGTC-3
, and
5
-GTGTC-3
. The rather loose specificity observed with the oligonucleotide templates is comparable to that observed with the phage
T4 (23, 24, 44) and E. coli (25, 26) primases on all
templates. In several other aspects, however, these three primases are
similar: (i) they contain a Cys4 type zinc motif (5, 22);
(ii) they require association with a helicase to achieve maximal
activity (28, 32-34, 45-47); (iii) they recognize a basic
trinucleotide sequence in which the 3
-nucleotide is cryptic (23-27);
and (iv) they synthesize relatively short oligoribonucleotides that
serve as primers for their respective DNA polymerases (23, 28-31, 44,
48, 49). On the other hand, they do differ in one fundamental manner.
Although all three primases require a helicase to translocate 5
to 3
in order to reach primase recognition sites, only the T7 63-kDa gene 4 primase has an inherent helicase activity (1, 13). Both the T4 gene 61 helicase and the E. coli DnaB helicase are encoded by
separate genes, and hence their respective primases must subsequently
associate with them to benefit from their translocation activities. As
a consequence the gene 4 primase catalyzes the synthesis of
oligoribonucleotides at a considerably faster rate (0.8 oligoribonucleotide/s/hexamer of protein on M13 ssDNA) than do either
the T4 gene 61 protein (0.83 × 10
2
oligoribonucleotide/s/monomer) (24) in the presence of helicase or the
E. coli DnaG protein (0.89 × 10
4
oligoribonucleotide/s/monomer) (39) in the absence of helicase.
We propose that the translocation activity of T7 primase is critical
for the acquisition of primase sites on large DNA molecules such as M13
ssDNA and that it also accounts for the greater specificity for the
primase sites 5-GGGTC-3
, 5
-TGGTC-3
, and 5
-GTGTC-3
on M13 DNA. In
the case of M13 ssDNA, primase sites are reached via the translocation
activity of the gene 4 protein, whereas sites on the considerably
shorter oligonucleotides are most likely accessed by random collision
with the template. In the former case we suggest that there is a
competition between the rapid translocation activity (300 nt/s) and the
synthesis of oligoribonucleotides (0.8 oligoribonucleotide/s) at a
primase recognition site. In this regard it is important to recall that
the gene 4 protein translocates 5
to 3
on the strand to which it is
bound but upon reaching a primase site polymerization of nucleotides
must proceed in the opposite direction, 3
to 5
with regard to the
template strand. As discussed below the mechanism of formation of the
first phosphodiester bond to yield pppAC may well differ from that
involved in the formation of subsequent phosphodiester bonds.
Consequently, when the protein is in a translocation mode it may not
linger for a sufficient time to extend the dinucleotide in those cases where a kinetically less favorable base pair is involved, namely the
incorporation of GMP or UMP. The fact that the incorporation of GMP at
the primase site 5
-GCGTC-3
decreases once the 5
-flanking region
exceeds a length of 20 nucleotides supports this interpretation. Below
this critical length the helicase domain does not have a sufficient
stretch to engage its 5
to 3
translocation activity thus providing
the gene 4 protein with sufficient time to incorporate an unfavorable
nucleotide.
Likewise, we believe that our finding (Fig. 7) that the
incorporation of GMP at the site 5-TCGTC-3
always results in
the synthesis of a functional tetraribonucleotide primer,
pppACGA, when primer synthesis is coupled to DNA synthesis is
consistent with the above interpretation. The intermediate pppACG
is not observed under these conditions but pppACGA is found at the
5
-termini of a significant number of Okazaki fragments. This finding
is compatible with a model in which the gene 4 protein incorporates GMP
at this site only when its translocation movement has slowed, an event
that also facilitates the transfer of the resulting oligoribonucleotide to the DNA polymerase for use as a primer.
Studies with E. coli DnaG primase indicate that the rate
determining step in primer synthesis is the formation of the first phosphodiester bond to generate a dinucleotide (39), thus suggesting that this reaction proceeds by a different mechanism than the subsequent extension of the dinucleotide. Cross-linking studies have
shown that the DnaG primase and -subunit of E. coli RNA polymerase bind ATP or GTP, the first nucleotide, and maintain contact
with it during the extension reaction of the next several nucleotides
(50, 51). At some point however, RNA polymerases must release the
contact between extending nucleotides and the starting nucleotide and
switch to a stable elongation mode. In those instances where an
efficient transition between the two modes does not occur, abortive
transcripts are released (52-54). This model of polymerization readily
explains our observation that misincorporation of nucleotides at a
primase recognition site is rare within the first three nucleotides but
occurs more frequently at the fourth and fifth nucleotide position
(Fig. 4). We propose that the lack of misincorporation in the first
three positions arises from a more stable interaction of the primase with the growing chain through simultaneous contacts with the starting
nucleotide and the 3
-end. The model of polymerization can also explain
the observation in Fig. 4 that oligoribonucleotides with GMP or UMP at
the third position of the primer were more efficiently extended to the
5
-terminus of the templates than were those with CMP at this position.
Incorporation of GMP or UMP at this position may require a switch from
the elongation mode to the stable phase and therefore the incorporation
of GMP or UMP leads to a complex that now readily polymerize nucleotide to the end of the template.
DNA primases and RNA polymerases lack a proof reading exonuclease characteristic of that found in many DNA polymerases. RNA polymerases, however, have a much higher fidelity of incorporation than do DNA primases (43). Since each event of primer synthesis involves a competition between the opposing movements of the associated helicase translocation activity and the polymerization of ribonucleotides, it seems reasonable that DNA primases have evolved such that they sacrifice fidelity for speed of primer synthesis. Furthermore, the resulting primers are eventually removed from the Okazaki fragments by RNase H activities in preparation for ligation.
We are grateful to Benjamin B. Beauchamp, Joonsoo Lee, Daochun Kong, and Stanley Tabor for providing purified proteins and helpful discussions. We also are very grateful to Khandan Baradaran for her comments and constructive criticisms on the manuscript.