(Received for publication, September 21, 1994; and in revised form, December 18, 1994)
From the
In the eukaryotic cell, DNA synthesis is initiated by DNA
primase associated with DNA polymerase . The eukaryotic primase is
composed of two subunits, p49 and p58, where the p49 subunit contains
the catalytic active site. Mutagenesis of the cDNA for the p49 subunit
was initiated to demonstrate a functional correlation of conserved
residues among the eukaryotic primases and DNA polymerases. Fourteen
invariant charged residues in the smaller catalytic mouse primase
subunit, p49, were changed to alanine. These mutant proteins were
expressed, purified, and enzymatically characterized for primer
synthesis. Analyses of the mutant proteins indicate that residues
104-111 are most critical for primer synthesis and form part of
the active site. Alanine substitution in residues Glu
,
Asp
, and Asp
produced protein with no
detectable activity in direct primase assays, indicating that these
residues may form part of a conserved carboxylic triad also observed in
the active sites of DNA polymerases and reverse transcriptases. All
other mutant proteins showed a dramatic decrease in catalysis, while
mutation of two residues, Arg
and Arg
,
caused an increase in K
.
Analysis of these mutant proteins in specific assays designed to
separately investigate dinucleotide formation (initiation) and
elongation of primer indicates that these two activities utilize the
same active site within the p49 subunit. Finally, mutations in three
active site codons produced protein with reduced affinity with the p58
subunit, suggesting that p58 may interact directly with active site
residues.
DNA primase initiates DNA replication by the synthesis of small
ribonucleotides called primers(1) . The mammalian primases are
composed of two subunits, p49 and p58, which purify as a complex
tightly bound to DNA polymerase
(2, 3, 4) . The tight association of
primase with DNA polymerase
implicates the DNA polymerase
as the lagging-strand DNA polymerase in
replication(1, 4) . In the in vitro SV40
replication system, DNA polymerase
elongates the RNA primer to
complete the synthesis of Okazaki fragment(5) . Okazaki
fragments are then extended by either DNA polymerase
or
,
allowing DNA polymerase
-primase to recycle and initiate another
Okazaki fragment on the lagging strand(6) . This essential role
of the DNA polymerase
-primase makes it a key component for
regulation and inhibition of the initiation of DNA replication.
One
unique property of primases (as well as RNA polymerases) is the ability
to synthesize nucleotides de novo on a template by the
formation of an initial dinucleotide. Primase initiates synthesis with
a triphosphate purine moiety at the
5`-end(7, 8, 9) . Relatively few errors are
made during the formation of the dinucleotide, whereas the primase
readily misincorporates ribonucleotides during elongation of this
dinucleotide(10, 11) . After synthesis of 7-10
ribonucleotides, the primer-template is translocated intramolecularly
to the active site of the DNA polymerase
subunit(12, 13) .
The mechanism of dinucleotide and
primer formation by primase is not well understood but may be similar
to the mechanism utilized by DNA polymerases. Even less is known about
the functional roles of specific amino acids within the primase
subunits. Superimposition of the derived polymerase crystal structures
of the Klenow fragment, human immunodeficiency virus, type 1 reverse
transcriptase, T7 RNA polymerase and DNA polymerase demonstrates
that, although there is little amino acid homology between such
diverged polymerases, the overall folding in the active sites is
conserved, bringing certain key residues into the correct position for
catalysis or substrate
binding(14, 15, 16, 17) . These key
residues are highly conserved in nearly all DNA and RNA polymerases and
reverse transcriptases, making up motifs A, B, and C (18) .
Central to these three motifs are the aspartic and glutamic acids,
which make up a catalytic triad within the structures(14) .
Mutagenesis experiments on DNA polymerases demonstrate the importance
of these residues in metal binding and
catalysis(19, 20) . Although the eukaryotic DNA
primases do not display any obvious amino acid sequence similarity to
motifs A, B, and C in the DNA polymerases, it is reasonable to propose
that the active site of the primases will fold similarly to those of
the DNA polymerases, reverse transcriptases, and T7 RNA polymerase.
Hence, it would be valuable to identify critical amino acid residues in
the DNA primase that may make up the equivalent of motifs A, B, and C.
This identification would also help to define structural elements
responsible for the low fidelity observed with the primases compared
with the higher fidelity of DNA polymerases.
Analysis of the recombinant mouse primase subunits demonstrates that both subunits are required for initiation while only the smaller subunit is necessary for elongation(12) . In contrast, the smaller subunit of the yeast primase complex appears to be sufficient for primer synthesis(21) . This discrepancy may be the consequence of the expression and purification procedures used or may represent a difference between species. Indeed, the mouse subunits do not complement temperature-sensitive and deletion alleles of the yeast primase genes(22) . Alternatively, it has been shown with both the yeast and mouse that the larger subunit stabilizes the activity of the p49 subunit(12, 21) . Perhaps, the higher mammalian primase subunits require this stabilization for initiation.
Amino acid alignment between the yeast (23) and the mouse p49 reveals a 34% identity, where the greatest similarity occurs in the N-terminal half of the two proteins(24) . This homology shows five regions of highly conserved amino sequences(24) . Several temperature-sensitive mutants in the primase of Saccharomyces cerevisiae were generated, and their mutations were determined to be in close proximity to or within these conserved regions(25) . Yeast cells carrying these mutant alleles have reduced primase activity and display a hyper-recombination and mutator phenotype, similar to other DNA replication mutants(26) .
On the basis of the homology between the yeast and mouse p49 primase subunits, we have generated a series of site-specific mutations in invariant charged amino acids in the mouse primase. Enzymatic characterization of these mutant proteins showed many of these charged residues to be critical for primer synthesis. We discuss the roles of these residues in the context of the two activities of primases, initiation and elongation of the primer.
Analysis of the amino acid sequence homology between the mouse, human, and S. cerevisiae catalytic primase subunit reveals five conserved regions (Fig. 1) containing numerous charged residues. Of these residues, 14 are found within clusters of conserved amino acids in regions IV and V(24, 30) . Fig. 1shows the locations of these residues within the p49 sequence. These 14 charged residues were changed to alanine to address their role in primer synthesis. Mutations were screened by dideoxy sequence analysis and by the presence of the new restriction site. Mutant and wild-type p49 proteins were expressed in and purified from Escherichia coli as histidine-tagged proteins (H-p49). Fig. 2shows a Coomassie-stained gel of the purified p49 mutants as well as the mouse p58 and human p180 subunits used throughout this study. These mutant proteins had the same apparent molecular weight as the wild-type p49 and were more than 90% homogeneous.
Figure 1: Schematic diagram of the locations of the alanine mutations in the linear amino acid sequence. Shown as boxes in the linear diagram are the five conserved regions in the p49 subunit(24) . Ticmarksbelow the boxes represent the location of the residues mutated in this study and the Cs above region IV represent Cys residues that lie in a putative zinc finger. In the lowerpart of the figure is the alignment of these regions from mouse, human, and yeast. Invariant residues are listed below the alignments, and boldfaced amino acids represent amino acids that were altered to alanine. The mouse p49 sequence is from (24) ; the human sequence is from (39) , and W. C. Copeland, unpublished observations. The yeast sequence is from (23) .
Figure 2:
SDS-PAGE analysis of mutant 49 subunits.
Coomassie Blue stained gel of the purified wild-type and mutant p49
subunits. Also shown are the mouse p58 subunit and single subunit human
DNA polymerase (p180) used in the assays throughout this work.
Approximately 0.5-2 µg of protein were loaded in each
lane.
Figure 3: Interaction of p58 with wild-type and mutant p49 subunits. Coomassie-stained gel containing co-precipitated GST-p58 protein with bound wild-type and mutant H-p49 subunits. Glutathione-Sepharose beads were mixed with preformed samples and washed as described under ``Experimental Procedures.'' Beads were then boiled in SDS sample buffer and loaded onto a 9% SDS-PAGE gel. Protein combinations that were mixed with the glutathione-Sepharose beads are listed on top of each lane of the gel. Lanes1-3 are control samples. GST-p49 (68 kDa) and H-p49 (49 kDa) were precipitated in lane1 to show that only the GST-p49 binds the glutathione-Sepharose beads. GST-p58 (82 kDa) was precipitated in lane2, while H-p49 was precipitated in lane3 with the glutathione-Sepharose beads to show nonspecific binding. Samples in lanes4-18 were co-precipitated with the GST-p58 protein where lane4 contains the wild-type p49. H-p49 signifies the histidine-tagged p49 protein while GST- signifies the glutathione S-transferase fusion proteins.
Figure 4: DNA binding assay. Elution profile of wild-type and E105A p49 subunits from single-stranded DNA cellulose. Shown are silver-stained gels of the eluted fractions from the wild-type (top) and E105A protein (bottom). The scale on the left shows the migration of the molecular weight standards. The heavyline represents the KCl concentration at that particular fraction according to the rightscale where the arrow represents 100 mM KCl.
Figure 5:
Coupled primase-polymerase assay on
poly(dT) and poly(dC). Relative activities of the mutant proteins and
wild-type primase complexes as measured in the coupled
primase-polymerase assay. Solidbars represent
activity with poly(dT) as template and
ATP/[-
P]dATP as label, while stripedbars represent activity with poly(dC) as the template and
GTP/[
-
P]dGTP as label. Results represent
the average of two or more experiments.
Figure 6:
Steady state kinetic analysis of primase
mutant proteins on poly(dC). A, autoradiogram of 15%
sequencing gel showing the de novo synthesis of primers on
poly(dC) template under increasing [-
P]GTP
concentrations. Names of the mutant proteins are listed below the lanes. Lanes1-7 represent
the primers formed with 10, 25, 50, 100, 250, 500, and 750 µM [
-
P]GTP, respectively. Lane0 represents no enzyme control with 750 µM GTP. Molecular weight markers on the left side were
determined from
P-end-labeled DNA oligonucleotides. B, bar graphs representing the individual kinetic parameters
as determined by PhosphorImager analysis of the reaction products in
gels. Only those mutant proteins that gave measurable product are
shown. Results represent the average of three or more
experiments.
Figure 7:
Analysis of dinucleotides formed by the
wild-type and mutant primase complexes. Reactions were performed as
described under ``Experimental Procedures.'' A,
dinucleotide products were treated with alkaline phosphatase and
separated on a 20% sequencing gel followed by autoradiography and
PhosphorImager analysis. Lane1 represents the
products with 250 µM GTP and 1.5 mM [-
P]ATP. Lane2 represents the products with 1.5 mM GTP and 1.5 mM [
-
P]ATP. Lane3 represents the products with 1.5 mM GTP and 250
µM [
-
P]ATP. Reactions in lane4 contained only 1.5 mM [
-
P]ATP as substrate. B,
graph showing the PhosphorImager quantitation of the dinucleotide
produced with 1.5 mM GTP and 1.5 mM [
-
P]ATP. Only those mutant proteins
that produced detectable dinucleotide are shown. Values shown are the
average of three or more experiments. The photograph of the
autoradiogram in A does not reflect the detection limit of
this assay and does not show the dinucleotide produced by the K104A
mutant.
Figure 8:
Extension of Oligo(A)-primed poly(dT) by
wild-type and mutant p49 proteins. Products from the reaction
containing oligo(A)-primed poly(dT),
[-
P]ATP and individual p49 proteins were
separated on a 15% sequencing gel and subjected to autoradiography as
described under ``Experimental Procedures.'' A,
photograph of autoradiogram; B, PhosphorImager quantitation of
gel containing oligo(A) extension products where products are
represented as percent incorporation relative to the wild-type. Results
represent the average of three or more
experiments.
We describe here the first analysis of the structure-function
relationships in the eukaryotic primase. Invariant charged amino acids
within highly conserved regions of the mouse p49 subunit were altered
to alanine to address their role in primer synthesis. These mutant
proteins were characterized on the basis of what is required for primer
synthesis. Previous characterization of the calf thymus primase and
overexpressed mouse primase subunits has demonstrated that in vitro primer synthesis by the eukaryotic primase can be divided into six
separate steps including two distinct enzymatic reactions: 1)
association of the p49 subunit with the p58 subunit; 2) binding of the
p49p58 complex to single-stranded DNA; 3) binding of the incoming
ribonucleotides to the primase-single-stranded DNA complex; 4)
initiation of primer synthesis by formation of a dinucleotide; 5)
elongation of the dinucleotide to make full-length primers; and 6) an
intramolecular switch of the full-length primer and template from the
primase active site to the DNA polymerase
active
site(12, 13, 33) .
The activities of these
mutant proteins were tested on a wide variety of substrates designed to
obtain steady-state kinetics parameters and to separately investigate
the initiation and elongation reactions of primer synthesis (Table 1). The E103A mutant behaved similarly to the wild-type
enzyme in most all assays. Hence, the importance and high conservation
of Glu are unclear. Perhaps more definitive experiments
are necessary to elucidate the function of Glu
. Three
mutant proteins, E105A, D109A, and D111A were the least active. E105A
and D109A proteins had only trace amounts of activity in the coupled
primase-polymerase assay using poly(dC), while D111A had no detectable
activity. However, these three mutant proteins displayed wild-type like
elution from single-stranded DNA cellulose and gave a similar digestion
pattern with thermolysin as a function of temperature, indicating that
the loss of activity was not due to any gross aberration in structure.
The D114A mutant demonstrated a dramatically altered substrate
preference in the coupled primase-polymerase assay relative to the
wild-type. The lower activity detected with poly(dT) in this assay by
D114A was unusual since a relatively high activity was observed in the
extension and initiation assays. Taken together, these data suggest
that the D114A mutant may have difficulty initiating de novo synthesis with poly(dT), while initiation with the (ATC) oligonucleotide may be more efficient due to the GTP requirement
at the 5`-end. This difference in initiation by the D114A mutant
suggests that Asp
may participate in the recognition of
the 5` purine residue.
Steady state kinetic analysis demonstrated
that the greatest effect observed with the active mutant proteins was
on k with only a slight change in apparent K
. The exceptions were the two
arginine mutant proteins at positions 162 and 163 in which the
5-10-fold increase in K
indicates a role in nucleotide binding. The positively charged
side chain of these arginines may bind the negatively charged phosphate
moiety of the incoming nucleotide. Lysine and arginine residues in
motif B of the Klenow fragment also have a similar
role(35, 36) . In addition, the major product formed
by these two arginine mutant proteins with poly(dC) was the 9-mer,
whereas all other active mutant proteins produced 15-20
nucleotide products. This may indicate a role for these arginine
residues in processivity.
The lack of activity in direct primase
assays with the E105A, D109A, and D111A mutant proteins suggests that
these residues play an essential role in primer synthesis. In DNA
polymerases, conserved aspartic and glutamic acid residues make up the
triad of negative charges in the active site and are critical for metal
binding and catalysis(14, 19, 20) . A similar
motif may exist in primase with Glu, Asp
,
and Asp
to make up part of the core active site. Given
the close proximity and affect of alanine changes at residues
Asp
and Asp
, it is tempting to speculate
that these two residues make up the equivalent of the two negative
charges found in motif C of the DNA polymerases. However, a clearer
definition of the role of these residues requires additional
experiments (analysis of mutant proteins that show partial activity).
The assignment of the other putative motifs, namely A and B, is
difficult to make at this time. There is no obvious homology between
motifs A, B, and C of the DNA polymerases (18) and the
eukaryotic primases, which is probably a consequence of the extreme
divergence of the primases from the polymerases. The recently
determined structures of the DNA polymerase (16, 17) demonstrate that these motifs are not always
co-linear. Thus, if residues 105-111 have a motif C-like
function, then it is quite possible that motif A or B is C-terminal to
this region and contains some of the residues mutated in this study.
Three mutant proteins, E105A, E148A, and D149A, demonstrated a reduced affinity with the p58 subunit. However, since neither wild-type (histidine-tagged or glutathione S-transferase-tagged) nor mutant proteins displayed any de novo primase activity in the absence of the p58 subunit, the primase activity observed by the E105A, E148A, and D149A mutant proteins in the coupled primase-polymerase assay do demonstrate an association with the p58 subunit under the lower salt assay conditions. Therefore, the apparent reduced affinity with p58 by the E105A, E148A, and D149A mutant proteins only occurred under the high salt conditions. The E148A and D149A mutant proteins were also interesting since they produced more product with poly(dT)/ATP than with poly(dC)/GTP in the coupled primase-polymerase assay. Surprisingly, these mutant proteins performed very poorly with oligo(A)-primed poly(dT).
It is noteworthy that these studies
identify two regions, 105 and 148-149, to interact with the p58,
especially since Glu is proximal to two other essential
amino acid side chains, Asp
and Asp
, and
probably deep in the active site. This suggests that the role of p58
during initiation may occur by interacting with active site residues.
The requirement of p58 in initiation and interaction with the active
site residues is very different than what would be expected of a DNA
polymerase. However, the architecture of a primase or RNA polymerase
must be somewhat different from a DNA polymerase since it is required
to hold two nucleotides in the active site instead of one during
initiation. Studies of two other RNA polymerases show the involvement
of other subunits or domains in initiation. Mutagenesis and structural
analysis of the T7 RNA polymerase implicates the N-terminal region
outside of the palm-fingers-thumb active site region in binding the
initiating ribonucleotide(15, 37) . More recently, it
was shown that a highly conserved region in the E. coli RNA
polymerase
factor cross-links the initiating nucleotide and
forms a face of the active site with the
subunit of the E.
coli RNA polymerase (38) . We previously demonstrated that
the p58 subunit of the mouse primase also weakly cross-links labeled
ATP(12) . Hence, the weaker affinity of the E105A protein to
bind the p58 subunit and the close proximity of Glu
within the active site suggest that, as with the
subunit
of RNA polymerase(38) , the p58 subunit may form a binding
surface for an initiating nucleotide within the active site.
For all the mutant proteins studied, we observed similar levels of activities between both the initiation and elongation assays as compared with the kinetic and coupled polymerase-primase assays. In general, these mutations failed to separate the two activities, which suggest a tightly coupled mechanism between the dinucleotide formation and elongation, possibly and most likely utilizing the same active site residues. In addition, the precise involvement of the p58 subunit in the initiation still remains obscure. Clearly a mutagenesis study in the larger subunit is warranted.