From the
Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric
helicase-primase composed of the products of the three DNA
replication-specific genes UL5, UL8, and UL52 (Crute, J. J., and Lehman, I. R.(1991) J. Biol. Chem. 266, 4484-4488). The
UL5 and UL52 products
constitute a heterodimeric subassembly of the holoenzyme that contains
both helicase and primase activities (Calder, J. M., and Stow, N.
D.(1990) Nucleic Acids Res. 18, 3573-3578; Dodson, M.
S., and Lehman, I. R.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1105-1109). The role of the
UL52 product in the
active HSV-1 helicase-primase was examined. A sequence located between
residues 610 and 636 on the UL52 protein was found to be conserved
among the UL52 homologues of eight herpesviruses. The carboxyl-terminal
portion of this conserved sequence consisted of two Asp residues
separated by a variable hydrophobic amino acid residue and is analogous
to the divalent metal-binding site of DNA polymerases and several DNA
primases. This motif has been designated the herpesvirus primase
DXD motif. To study the role of the HSV-1 primase DXD
motif in primase action, three site-directed changes were introduced
into the UL52 gene. The helicase activity of the recombinant
holoenzymes was unaffected by any of the introduced changes. Changing
either of the two Asp residues that constitute the divalent
metal-binding site (Asp
Herpes simplex virus type 1 (HSV-1)
The predicted amino acid sequence
of the UL5 product has been found to contain seven conserved
amino acid sequence motifs typical of superfamily I of confirmed or
putative helicases
(4) . This suggests that the UL5 product functionally contributes to the helicase activity of the
holoenzyme. The predicted amino acid sequence of the UL52 protein is
not homologous to any known gene family.
The native size and distribution of the products synthesized in the
coupled assay were also visualized. Reactions were performed as
described above for the radiolabeled coupled assay, except that
[
The other type of indirect primase assay examined the
native size and distribution of products synthesized on
We have focused our current studies on the primase of the
HSV-1 helicase-primase enzyme complex. In performing multiple sequence
alignments of UL52 homologues from eight different herpesviruses, we
delineated several conserved regions. One of the regions contained two
conserved Asp residues in the predicted HSV-1 UL52 amino acid sequence
at positions 628 and 630. The two Asp residues were contained within a
locally hydrophobic region that resembled the putative divalent
metal-binding site identified in many DNA polymerases and primases. We
have designated the conserved structure the herpesvirus primase
DXD motif. Because of the similarity between the herpesvirus
primase DXD motif and the DNA polymerase metal-binding site,
in addition to the presence of this motif on other identified primases,
the role of this structure in the HSV-1 helicase-primase holoenzyme was
explored. Specific changes were introduced into the UL52 sequence by site-directed mutagenesis, and the resultant HSV-1
helicase-primase holoenzymes were analyzed.
In helicase assays
performed on the altered HSV-1 helicase-primase holoenzyme
[UL52(D628A)], [UL52(D630A)], or
[UL52(N624G)], ATP-dependent unwinding was nearly the same as
in the wild-type enzyme. The slight differences observed may reflect
variability in enzyme preparations rather than intrinsic changes in
enzyme activity. More interestingly, profound differences were observed
in the ability of the enzymes to synthesize RNA primers. In either
indirect or direct primase assays, alteration of either of the Asp
residues in the HSV-1 primase DXD motif (Asp
Therefore, at least a part of the primase active site of the HSV-1
helicase-primase is contained within the UL52 product of the
heterotrimeric holoenzyme. The two Asp residues contained within the
HSV-1 primase DXD motif on the UL52 protein were found to be
essential for in vitro and presumably in vivo RNA
primer synthesis. Using a strategy of mutating conserved charged
residues in the UL52 protein, Klinedinst and Challberg
(25) have
also concluded that the UL52 product is the primase subunit of
the HSV-1 helicase-primase holoenzyme. By analogy to the similar motif
in conserved region I of the eukaryotic DNA
polymerases
(21, 22, 24) , we tentatively propose
that the critical function of the herpesvirus primase DXD
motif is to bind NTP by coordination to nucleotide-bound
Mg
Primer synthesis may not
be the only function provided by the HSV-1 UL52 protein in the
helicase-primase. Although in the other studied helicase-primase
systems subunits specific for primase and helicase activities have been
delineated (e.g. T4 gene 41/61, T7 gp4/gp4*, and E. coli dnaB/dnaG helicase-primases)
(26) , the herpesvirus
helicase-primases have not been examined sufficiently for this
conclusion to be drawn. Additionally, since activities involved in
primase function, NTP interaction, and DNA binding are also required
for DNA unwinding, functional overlap between RNA primer formation and
duplex DNA unwinding may occur. Furthermore, ``zinc finger''
motifs are thought to be essential for the sequence-preferred priming
activity demonstrated for several prokaryotic primases
(27) .
Interestingly, within the herpesvirus UL52 homologues, there also
exists a conserved zinc finger domain at the extreme carboxyl-terminal
part of the protein.
Primase assays were performed as
described under ``Materials and Methods'' for the indirect
assay. Assays were initiated by the addition of the indicated HSV-1
helicase-primase holoenzyme and incubated for 1.0 h. One unit of
primase activity incorporates 1.0 pmol of dNTP into polymeric DNA/h.
Background incorporation values of 20.2 and 5.4 pmol were obtained in
the absence of added helicase-primase for Sequenase and the HSV-1 DNA
polymerase holoenzyme, respectively. These were subtracted to obtain
the results presented.
We are grateful to many of our colleagues for aid
during the course of this investigation. Drs. Mark Dodson, Thomas
Hernandez, and Robert Lehman generously provided the HSV-1
UL5, UL8, UL30, and UL52 recombinant baculoviruses. Drs. Paul Olivo and Mark Challberg made
available the HSV-1 UL42-containing plasmid pNN4 and Dr. David
Knipe the HSV-1 UL29-containing plasmid p8-BS. Drs. Daniel
Tenney and Robert Hamatake made available details of work in progress
on the primase of the HSV-1 helicase-primase. We also wish to extend
our appreciation to Susan Goldrick for constructing the UL42 and UL29 recombinant baculoviruses, maintaining viral
stocks, and overexpressing several of the reagents used here.
Additionally, we thank Dr. Eugene McNally and Paul McGoff for helpful
suggestions used in the development of scaled procedures for the
isolation the HSV-1 replication proteins and Dr. Robert Eckner for
assembly of the helicase substrate.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or Asp
) to Ala
dramatically reduced the primase activity of the HSV-1 helicase-primase
holoenzyme in vitro, whereas alteration of the nearby
conserved residue Asn
to Gly had minimal effect.
Therefore, in the three-subunit HSV-1 helicase-primase, the UL52 product provides at least a part of the primase catalytic site.
(
)
contains seven nondispensable DNA replication-specific
genes
(1) . These seven genes encode four functionally different
DNA replication proteins or protein complexes. A homodimeric
origin-binding protein helicase is encoded by UL9, a monomeric
SSB is encoded by UL29, and a heterodimeric DNA polymerase is
encoded by UL30 and UL42. The products of the
remaining three DNA replication genes, UL5, UL8, and
UL52, constitute a heterotrimeric helicase-primase. The
UL5 and UL52 products have been shown to form a
stable isolable subassembly of the HSV-1
helicase-primase
(2, 3) . This complex contains all of
the measurable enzymatic activities associated with the holoenzyme. The
UL8 product does not seem to participate directly in the
catalysis of either DNA unwinding or RNA primer synthesis. Isolation of
the individual UL5 and UL52 products has shown that
neither protein alone has appreciable helicase activity; preparations
of the UL52 protein have been found to contain low levels of primase
activity.
(
)
(
)
We
have now examined the function of the UL52 product in the
active HSV-1 helicase-primase by a combination of sequence comparisons
and site-directed mutagenesis. Multiple sequence alignment of the
predicted amino acid sequence of the UL52 product with seven
other identified herpesvirus UL52 homologues delineated
several conserved regions. One of these resembled the putative
metal-binding site found in prokaryotic and eukaryotic primases and
eukaryotic DNA polymerases. This conserved region contained two Asp
residues and was designated the herpesvirus primase DXD motif.
To explore the function that this site provides in the HSV-1
helicase-primase, we introduced three point mutations into the
DXD motif of the UL52 sequence. Recombinant
helicase-primase holoenzymes were then expressed in insect cells,
isolated, and studied in vitro by comparison to the wild-type
enzyme. From this analysis, we have tentatively concluded that the
UL52 product contains at least part of the primase catalytic
site in the HSV-1 helicase-primase holoenzyme.
Reagents
All reagents were of the highest
quality available. Linearized baculovirus DNA and the baculovirus
transfer vector pVL1393 were from Invitrogen; BaculoGold DNA was from
Pharmingen. Grace's medium was obtained in powdered form from JRH
Biosciences. Pluronic F-68 was from Life Technologies, Inc. Sf9 cells
were from the American Type Culture Collection; Sf21 cells were from
Invitrogen. Source 15Q was obtained from Pharmacia Biotech Inc. in bulk
form and packed as an 8-ml column (1.0 10 cm) using a fast
protein liquid chromatography system. A prepacked column of Sephacryl
S-300 HR (1.6
60 cm; Pharmacia Biotech Inc.) was used for gel
filtration. The plasmid pBluescript II SK
was from
Stratagene. Modified T7 DNA polymerase (Sequenase) was from United
States Biochemical Corp.
X174 single-stranded DNA was from New
England Biolabs Inc. Radiolabeled reagents were from DuPont NEN.
Oligonucleotides used as size standards were from Pharmacia Biotech
Inc.
Amino Acid Sequence Analysis
Amino acid sequences
were obtained from the GenBank Data Bank (Release 74.0).
Data base searches for sequence similarity were performed using
programs based on the BLAST algorithm
(5) and the BLOSUM62
matrix for comparison of amino acid residues
(6) . Multiple
alignments of amino acid sequences were generated using the MACAW
programs
(7) . Data base searching for sequence motifs was
performed using the programs DBSITE and FPAT (National Center for
Biotechnology Information, National Institutes of Health).
Buffers
Buffer A was 20 mM
NaHEPES, pH 7.5, 1.0 mM dithiothreitol, 10
µg/ml leupeptin, 10 µg/ml pepstatin A, 10 mM sodium
bisulfite, pH 7.6, and 1.0 mM phenylmethylsulfonyl fluoride.
Buffer B was Buffer A to which was added 10% (v/v) glycerol, 200
mM NaCl, and 2.0 M ammonium sulfate; sodium bisulfite
was omitted. Buffer C was Buffer A to which was added 10% (v/v)
glycerol, 1.0 mM EDTA, and 1.0 mM EGTA with sodium
bisulfite deleted. Buffer D was 20 mM Tris-HCl, pH 8.0, 10%
(v/v) glycerol, 1.0 mM dithiothreitol, 5.0 mM
MgCl
, and 200 µg/ml acylated bovine serum albumin.
Buffers were filtered through 0.22-µm membranes and degassed by
sonication prior to use.
Cells, Viruses, and DNA
Routine methods were used
throughout for the production of DNA constructs and recombinant
baculoviruses
(8, 9) . Spodoptera frugiperda cells (Sf9 or Sf21) were maintained in suspension culture at 27
°C with shaking in Grace's medium supplemented with 0.33% TC
Yeastolate, 0.33% lactalbumin hydrolysate, 0.10% Pluronic F-68, and 10%
heat-inactivated fetal bovine serum
(10) . When Sf21 cultures
were scaled to the 1-liter level, gentamicin sulfate (50 µg/ml) was
added. The recombinant baculoviruses AcMNPV/UL5, AcMNPV/UL8,
AcMNPV/UL30, and AcMNPV/UL52
(11, 12) were from Drs.
Mark Dodson (University of Arizona), Thomas Hernandez (Stanford
University), and Robert Lehman (Stanford University). AcMNPV/UL29 was
constructed as follows. The RsrII-EarI fragment
between HSV-1 coordinates 62069 and 58259
(13) was removed from
the UL29-containing plasmid p8-BS (provided by Dr. David
Knipe, Harvard University) and inserted into the transfer vector
pVL1393 utilizing EcoRI linkers to yield pVL1393/UL29. The
correct insert was confirmed by nucleotide sequencing across the start
site of the transfer vector. AcMNPV/UL29 was isolated by several rounds
of plaque purification from the virus stock generated by cotransfection
of pVL1393/UL29 with linearized baculovirus DNA into Sf9 cells.
AcMNPV/UL42 was constructed as follows. Uracil-rich single-stranded DNA
was generated from the plasmid pNN4 (provided by Dr. Paul Olivo
(Washington University) and Dr. Mark Challberg (National Institutes of
Health)) in Escherichia coli strain CJ236 by infection with
M13KO7
(14) . An additional BamHI restriction site was
introduced by oligonucleotide-directed mutagenesis at HSV-1 coordinate
92094
(13) . This was termed pNN4/Bam. The fragment from the
introduced BamHI site to the unique FspI site in the
UL42 open reading frame was removed from pNN4/Bam, assembled
with the balance of the UL42 open reading frame removed from
pNN4, and ligated into BamHI-EcoRI-digested
pBluescript II SK to create pBS2/UL42. The full-length
UL42 open reading frame contained in the BamHI-EcoRI
fragment was removed and inserted into pVL1393 to construct
pVL1393/UL42. The identity of the construct was confirmed by nucleotide
sequencing. The recombinant baculovirus AcMNPV/UL42 was obtained as
described for AcMNPV/UL29. Virus stocks used for the production of
recombinant protein were generated in Sf21 cells, and their titer was
determined by plaque assay on Sf9 cells.
Construction of Expression Vectors for Altered UL52
Genes
Three site-directed mutations were introduced into the
UL52 open reading frame. The UL52 coding region was
removed from pVL941/UL52 (provided by Drs. Mark Dodson and Robert
Lehman) by BamHI digestion and inserted into pBluescript II
SK to create pBS2/UL52. Uracil-rich single-stranded
DNA was obtained as described for pNN4 and used as template in three
separate oligonucleotide-directed mutagenesis reactions. These changed
UL52 codons for Asn
, Asp
, and
Asp
to Gly
, Ala
, and
Ala
, respectively. These constructs were termed
pBS2/UL52(N624G), pBS2/UL52(D628A), and pBS2/UL52(D630A), respectively.
The 540-base pair NcoI-NcoI fragment was then removed
from each of the three constructs and reinserted into the parent
pBS2/UL52 construct lacking the identical fragment to give the
constructs pUL52(N624G), pUL52(D628A), and pUL52(D630A), respectively.
The full-length altered UL52 open reading frames were then transferred
to pVL1393 by BamHI digestion to create pVL1393/UL52(N624G),
pVL1393/UL52(D628A), and pVL1393/UL52(D630A), respectively. The
identity of each construct was then confirmed by nucleotide sequencing.
The recombinant baculoviruses AcMNPV/UL52(N264G), AcMNPV/UL52(D628A),
AcMNPV/UL52(D630A) were then obtained by cotransfection with the
respective transfer plasmids as described for the isolation of
AcMNPV/UL29, except that BaculoGold DNA was substituted for the
linearized baculovirus DNA.
Expression of Wild-type and Altered HSV-1
Helicase-Primase Holoenzymes
Sf21 cells (3.0 liter) were grown
to a density of 0.8-1.0 10
cells/ml,
collected by centrifugation, and infected by resuspension in 0.1
culture volume of a viral inoculum calculated to yield a multiplicity
of infection of 5-10 plaque-forming units of the indicated
viruses/cell. When necessary, the volume was adjusted with the medium
used for cell growth. The HSV-1 helicase-primases were expressed by
triply infecting the Sf21 cells with AcMNPV/UL5, AcMNPV/UL8, and
AcMNPV/UL52, AcMNPV/UL52(N624G), AcMNPV/UL52(D628A), or
AcMNPV/UL52(D630A) to obtain the HSV-1 helicase-primase, HSV-1
helicase-primase [UL52(N624G)], HSV-1 helicase-primase
[UL52(D628A)], or HSV-1 helicase-primase
[UL52(D630A)], respectively. After allowing 1 h for viral
adsorption, the cells were diluted to their original density with fresh
medium, and protein expression was allowed to proceed for 60-68
h.
Preparation of Cell Extracts and Ammonium Sulfate
Precipitation of HSV-1 Helicase-Primases
All procedures were
performed at 4 °C unless indicated. The baculovirus-infected Sf21
cells were harvested by centrifugation, washed once with Grace's
medium
(9) , and resuspended in 7 volumes of Buffer A. ATP and
MgCl were added to concentrations of 1.0 and 2.0
mM, respectively. Typical yields of cells were 7.0-10
g/liter. After incubation on ice for 10 min, the cells were lysed by
Dounce homogenization (15-20 strokes with a tight fitting
pestle), and the nuclear fraction was separated from the cytosolic
fraction by centrifugation (1000
g, 15 min). Cytosolic
fractions were retained and further clarified by ultracentrifugation
for 75 min at 100,000
g. HSV-1 helicase-primases were
precipitated with ammonium sulfate by mixing 1 volume of the
postmicrosomal supernatant solution with 1 volume of Buffer B and
incubating for at least 2 h on ice. The precipitates were harvested by
centrifugation and resuspended in a minimal volume of Buffer C
containing 200 mM NaCl. The conductivities of the samples were
determined, and the samples were diluted with Buffer C to a
conductivity corresponding to Buffer C containing 100 mM NaCl.
The diluted extracts were then clarified by ultracentrifugation
(440,000
g, 10 min).
Additional Purification of HSV-1
Helicase-Primases
The HSV-1 helicase-primases were purified from
ammonium sulfate-precipitated cytosolic extracts by chromatography
through Source 15Q and gel filtration through Sephacryl S-300 HR. The
diluted clarified fraction was loaded onto the Source 15Q column
equilibrated in Buffer C containing 100 mM NaCl; the column
was washed with 1.5 column volumes of the equilibration buffer; and the
protein was eluted with an 8-column volume linear gradient between
Buffer C containing 100 mM NaCl and Buffer C containing 800
mM added NaCl. Fractions containing the HSV-1 helicase-primase
eluted at a point in the gradient corresponding to 250 mM NaCl
and were identified by the presence of DNA-dependent ATPase activity
and the concomitant elution of the three polypeptides (M 120,000, 97,000, and 70,000) that form the HSV-1 helicase-primase
holoenzyme. Active fractions were further fractionated by gel
filtration through Sephacryl S-300 HR equilibrated in Buffer C
containing 300 mM added NaCl. The HSV-1 helicase-primase
eluted at a point in the chromatogram corresponding to ferritin
(M
440,000). Protein concentrations of pooled
fractions were determined utilizing a molar extinction coefficient at
280 nm of 269,000 M
cm
(15) . The enzyme was frozen in
aliquots under liquid nitrogen and stored at -80 °C. Between
15 and 25 mg of nearly homogeneous enzyme was obtained from each
preparation at a concentration of at least 1 mg/ml.
Isolation of the HSV-1 SSB and HSV-1 DNA Polymerase
Holoenzyme
For the HSV-1 SSB, the baculovirus AcMNPV/UL29 was
used to infect 6.0 liters of Sf21 cells. The isolation procedure was
identical to that for the HSV-1 helicase-primase holoenzyme, except
that ATP and MgCl were not added at the cell lysis step and
the Source 15Q column volume was increased to 18 ml (1.6
9.0
cm). Two gel filtration runs were performed to accommodate the
increased preparation size. For isolation of the HSV-1 DNA polymerase
holoenzyme, Sf21 cells (6.0 liters) were doubly infected with
AcMNPV/UL30 and AcMNPV/UL42. Lysates were prepared as described for the
HSV-1 SSB. The enzyme was initially precipitated with Buffer B
containing 2.5 M ammonium sulfate. The HSV-1 DNA polymerase
was isolated as indicated for the HSV-1 SSB. Purified protein samples
were quantitated with molar extinction coefficients at 280 nm of 70,400
and 114,000 M
cm
for the
HSV-1 SSB and HSV-1 DNA polymerase holoenzyme,
respectively
(15) . 440 mg of nearly homogeneous HSV-1 SSB
(10-15 mg/ml) and 20 mg of
95% pure HSV-1 DNA polymerase
holoenzyme (1.2 mg/ml) were obtained.
DNA Helicase Assays
DNA helicase assays were
performed with minor modifications as described using the double-tailed
substrate (16). The [-
P]ATP used to
radiolabel the 68-mer oligonucleotide was changed to
[
-
P]ATP. After gel filtration, the helicase
substrate was concentrated by membrane diafiltration. Additionally, the
concentration of the helicase substrate used in the unwinding reaction
was increased to 50 µM (in nucleotide). Quantitation of
the individual helicase reactions was as described
(17) .
Coupled DNA Primase-DNA Polymerase Assays
Coupled
DNA primase-DNA polymerase assays were performed essentially as
described (18, 19). Primase-dependent DNA polymerase activity was
assayed with the HSV-1 helicase-primase holoenzymes using either
modified T7 DNA polymerase (Sequenase) or the HSV-1 DNA polymerase
holoenzyme as the accessory DNA polymerase. Assays (25 µl) were
assembled on ice in Buffer D with 1.0 µg of X174
single-stranded DNA, 5.8 pmol of HSV-1 SSB, 1.0 mM ATP, 1.0
mM GTP, 100 µM CTP, and 100 µM UTP.
The three deoxynucleoside triphosphates dTTP, dCTP, and dGTP were added
at a final concentration of 50 µM.
[
H]dATP was included at 20 µM at a
final specific activity of 10 Ci/mmol. The indicated DNA polymerase was
added (3.0 units of modified T7 DNA polymerase (Sequenase) or 2.7 pmol
of HSV-1 DNA polymerase holoenzyme), and the reaction was initiated by
the addition of 3.4 pmol of the indicated HSV-1 helicase-primase
holoenzyme. One unit of DNA primase activity resulted in the
incorporation of 1.0 pmol of dATP into polymeric DNA/h at 34 °C.
H]dATP was omitted and 50 µM dATP
was included. Reactions were terminated by the addition of EDTA to 10
mM, phenol/chloroform/isoamyl alcohol (24:24:1)-extracted,
ethanol-precipitated, resuspended in 10 µl of a glycerol-containing
loading buffer, and electrophoresed through a 0.8% agarose gel. The
products of the coupled DNA primase-DNA polymerase reaction were
visualized by UV transillumination after staining with ethidium
bromide.
Direct DNA Primase Assays
Direct primase assays
were performed as described
previously
(18, 19, 20) . Reactions (25 µl)
were assembled as described for the coupled assay with the omission of
DNA polymerase and dNTPs. To visualize the products generated during
the course of the primer synthesis reaction, 10 µCi of
[-
P]UTP was included in addition to the
other three NTPs. Primer synthesis reactions were initiated by the
addition of 3.4 pmol of wild-type or mutant HSV-1 helicase-primase
holoenzyme. After incubation for 1.0 h at 34 °C, reactions were
terminated by the addition of formamide to 50% (v/v) and heating to 95
°C. Products of the primase reaction were separated from the
reactants by electrophoresis through 18% polyacrylamide gels containing
7.0 M urea as described
(8) . Size standards of known
chain length were radiolabeled with T4 polynucleotide kinase and
[
-
P]ATP. These were included in parallel
lanes as reference markers. After electrophoresis, the gel was fixed
with 10% trichloroacetic acid and dried under vacuum, and the products
of the primase reaction were visualized by autoradiography. The ability
of the HSV-1 DNA polymerase to utilize the RNA primers synthesized in
the direct primase reaction was also analyzed. For these experiments,
the assay components were assembled as described for the direct gel
assay. After the primer synthesis reaction, 2.7 pmol of HSV-1 DNA
polymerase holoenzyme and a 50 µM concentration of each of
the four dNTPs were added, and the reaction was allowed to proceed for
an additional 1.0 h. The products of the assay were processed as
described for the direct primase assay.
Identification of the Herpesvirus Primase DXD
Motif
Amino acid and nucleotide sequence data base searches with
the predicted HSV-1 UL52 amino acid sequence revealed significant
similarity (probability of matching by chance alone below
10) only to the homologous proteins of other
herpesviruses. Alignment of the predicted amino acid sequences of eight
herpesvirus UL52 homologues showed several conserved blocks.
Particularly noticeable was a conserved region of variable
hydrophobic-hydrophilic character contained within the UL52 homologues.
This region corresponded to residues 607-634 on the predicted
UL52 amino acid sequence (Fig. 1). The carboxyl-terminal
one-third of this region contained two conserved Asp residues (UL52
Asp
and Asp
) within a local group of
hydrophobic amino acid residues. This structure, designated as the
herpesvirus primase DXD motif, is similar to part of the
active center of family B DNA polymerases (e.g. region I) and
also to one of the conserved motifs found in prokaryotic and eukaryotic
primases
(21, 22, 23) .
Figure 1:
Alignment of the putative metal-binding
sites in primases, DNA polymerases, and UL52-related proteins:
identification of the herpesvirus primase DXD motif. Multiple
sequence alignments of the four groups of proteins were generated by
the MACAW program, and the conserved motifs were superimposed manually.
A, prokaryotic DNA primases; B, eukaryotic primases;
C, DNA-dependent DNA polymerases and terminal
deoxynucleotidyltransferases; D, herpesvirus UL52 homologues.
A-C depict the short amino acid residue stretches
containing the ``DXD'' motif; D shows the
complete block delineated by the MACAW program for the herpesvirus UL52
homologues. The consensus sequence, derived separately for each group,
consists of amino acid residues that are conserved in all sequences
(upper-case letters) or in all but one sequence
(lower-case letters). Consensus abbreviations are as follows:
U, bulky aliphatic residue (I, L, V, or M); &,
bulky hydrophobic residue (I, L, V, M, F, Y, or W); and ., any residue.
The two conserved aspartic acid residues that may directly interact
with the metal cation are in boldface type. For each protein,
the position of the first aligned residue in the sequence is indicated;
the numbers in parentheses are for partial sequences.
Each sequence is accompanied by the SwissProt, PIR, or GenBank
accession number. B.subt., Bacillus subtilis;
B.aphi., Buchnera aphidicola; R.prow.,
Rickettsia prowazekii; POL-beta, polymerase ;
TDT, terminal deoxynucleotidyltransferase; VZV,
varicella-zoster virus; HCMV, human cytomegalovirus;
MCMV, murine cytomegalovirus; HHV6, human herpesvirus
6; EHV, equine herpesvirus; EBV, Epstein-Barr virus;
HVS, Herpesvirus saimiri.
Production of UL52-mutated HSV-1 Helicase-Primase
Holoenzymes
We engineered three different alterations in the
HSV-1 UL52 sequence such that the UL52 protein was changed in
the domain that contains the HSV-1 primase DXD motif. Residues
Asp and Asp
were changed to Ala, and the
nearby conserved residue Asn
was changed to Gly.
Recombinant baculoviruses were then constructed (AcMNPV/UL52(D628A),
AcMNPV/UL52(D630A), and AcMNPV/UL52(N624G)) that expressed the UL52
protein with the respective alterations. Altered HSV-1
helicase-primases were individually expressed in insect cells triply
infected with baculoviruses recombinant for wild-type UL5,
wild-type UL8, and one of the altered UL52 genes. The
HSV-1 helicase-primases [UL52(N624G)],
[UL52(D628A)], and [UL52(D630A)] were isolated
according to a rapid purification scheme, and their in vitro properties were determined and compared with identically prepared
wild-type HSV-1 helicase-primase.
Helicase Activity Is Normal in the UL52-mutated HSV-1
Helicase-Primase Holoenzymes
The helicase activity of the HSV-1
helicase-primase holoenzyme was compared with that of the enzymes
containing the UL52 product mutations UL52(D628A),
UL52(D630A), and UL52(N624G). When the individual enzymes were assayed
for associated helicase activity, the three altered helicase-primase
holoenzymes were found to contain helicase activity nearly identical to
the wild-type HSV-1 helicase-primase (Fig. 2). The differences
observed in helicase activity were 15% of the wild-type enzyme
activity.
Figure 2:
Helicase activity of the HSV-1
helicase-primase holoenzyme is unaffected by mutations introduced into
the UL52 subunit. Helicase activities associated with the wild-type and
UL52-mutated HSV-1 helicase-primase holoenzymes were analyzed as
described under ``Materials and Methods.'' A,
lanes 1-5, 6-10, 11-15,
and 16-20 represent groups of helicase assays performed
with the HSV-1 helicase-primase holoenzymes containing wild-type UL52,
UL52(D628A), UL52(D630A), or UL52(N624G), respectively. Each set of
five assays was assembled to contain increasing amounts of the
indicated HSV-1 helicase-primase holoenzyme: 0.0, 1.7, 3.4, or 5.1
pmol. The final lane of each group contained 5.1 pmol of the indicated
HSV-1 helicase-primase holoenzyme with the omission of ATP from the
assay. The position of the electrophoretic origin of the gel
(origin) and the point of migration of the dehybridized
radiolabeled oligonucleotide product (68-mer) of the helicase
reaction are indicated. B, shown is the quantitation of
helicase activity. The activity of individual helicase reactions
outlined in A was quantitated as described under
``Materials Methods.'' The helicase activity of the
individual HSV-1 helicase-primase holoenzymes was as follows: ,
wild-type;
D1, UL52(D628A);
D2,
UL52(D630A);
N, UL52(N624G).
DNA Synthesis Initiated by the Primase of the HSV-1
Helicase-Primase Holoenzyme Requires an Intact Herpesvirus Primase
DXD Motif
Two types of indirect DNA primase assays were
performed with the wild-type and UL52-mutated HSV-1 helicase-primase
holoenzymes. Each assay examined the coupling of the primase activity
of the indicated HSV-1 helicase-primase holoenzyme to the DNA synthetic
activity of a heterologous or homologous DNA polymerase (Sequenase or
the HSV-1 DNA polymerase holoenzyme). In assays performed using a
radiolabeled dNTP to monitor coupled DNA primase-DNA polymerase DNA
synthesis, we found that the alteration of either Asp or
Asp
to Ala in the UL52 protein [UL52(D628A)] or
[UL52(D630A)] reduced holoenzyme priming activity to near
background levels (). Alteration of the conserved
Asn
in the UL52 product [UL52(N624G)]
had a minimal effect on the priming activity of the isolated
holoenzyme. Moreover, primase activity was independent of the DNA
polymerase used in the coupled assay (compare values obtained with
Sequenase with results obtained with the HSV-1 DNA polymerase
holoenzyme).
X174
single-stranded DNA (Fig. 3). Native agarose gel electrophoretic
analysis of the products synthesized in the indirect primase assay
showed that alteration of either Ala
or Ala
in the UL52 product [UL52(D628A)] or
[UL52(630A)] reduced the coupled DNA synthetic activity of
the holoenzyme to background levels. Minimal full-length replicative
form DNA was generated in the synthesis reaction when Sequenase was
used as the coupling DNA polymerase (Fig. 3, compare lanes2 and 3 with lane5). When the
HSV-1 DNA polymerase was used to couple RNA primer synthesis to DNA
chain elongation, slightly greater amounts of replicative form products
were noted when compared with the addition of DNA polymerase alone
(Fig. 3, compare lanes7 and 8 with
lane10). Furthermore, analysis of the HSV-1
helicase-primase holoenzyme [UL52(N624G)] showed near
wild-type priming levels with either DNA polymerase used (Fig. 3,
compare lanes4 and 9 with lanes1 and 6).
Figure 3:
DNA synthesis coupled to RNA priming by
the HSV-1 helicase-primase holoenzyme requires an intact herpesvirus
primase DXD motif. Coupled DNA primase-DNA polymerase assays
were performed as described under ``Materials and Methods''
for the indirect agarose gel-based assay. Lanes 1-5,
Sequenase was used as the coupled DNA polymerase; lanes
6-10, the HSV-1 DNA polymerase holoenzyme was used as the
coupled DNA polymerase. The primase used in individual assays was as
follows: lanes1 and 6, HSV-1
helicase-primase holoenzyme (wild-type); lanes2 and
7, HSV-1 helicase-primase holoenzyme [UL52(D628A)];
lanes3 and 8, HSV-1 helicase-primase
holoenzyme [UL52(D630A)]; lanes4 and
9, HSV-1 helicase-primase holoenzyme [UL52(N624)].
Assays in lanes5 and 10 contained no added
helicase-primase. Lanes M contained molecular size markers of
known length in kilobase pairs (kbp). Lane ss contained the input single-stranded DNA substrate. Lane ds contained X174 replicative form I (faster migrating band)
and II (slower migrating band) double-stranded DNAs to indicate the
position of migration of the fully duplex product of the
primase-polymerase reaction.
The HSV-1 Primase DXD Motif Is Essential for Primer
Synthesis by the HSV-1 Helicase-Primase Holoenzyme
The size
distribution and utilization of primers synthesized by wild-type and
altered HSV-1 helicase-primase holoenzymes were examined directly. The
HSV-1 helicase-primase holoenzyme and the HSV-1 helicase-primase
[UL52(N624G)] both synthesized primers of 12 nucleotides in
length (Fig. 4, lanes1 and 4,
respectively). Some RNA products of shorter chain length were also
seen. In addition, the autoradiograph showed approximately the same
amount of total RNA synthesized by each enzyme (Fig. 4, compare
lanes1 and 4). The full-length primers
synthesized by either the HSV-1 helicase-primase holoenzyme or the
HSV-1 helicase-primase holoenzyme [UL52(N624G)] were utilized
when the HSV-1 DNA polymerase holoenzyme and the four dNTPs were
included in the reaction (note transfer of radiolabeled material of 12
nucleotides in size to the upper part of the autoradiogram in
Fig. 4
, lanes6 and 9). In contrast,
the mutant HSV-1 helicase-primase holoenzyme [UL52(D628A)] or
[UL52(D630A)] contained negligible RNA primer synthetic
activity (Fig. 4, lanes2 and 3,
respectively). By densitometric scanning (Fig. 4, lanes
1-5), the difference in activity between the
primase-proficient (wild-type or [UL52(N624G)]-altered HSV-1
helicase-primase holoenzyme) and the primase-deficient (HSV-1
helicase-primase holoenzyme [UL52(D628A)] or
[UL52(D630A)]) enzymes was estimated to be at least 100-fold.
Figure 4:
The herpesvirus primase DXD motif
is essential for the synthesis of RNA primers by the HSV-1
helicase-primase holoenzyme. Direct primase assays were performed as
described under ``Materials and Methods.'' The primase used
in the individual assays was as follows: lanes 1 and
6, HSV-1 helicase-primase holoenzyme (wild-type); lanes2 and 7, HSV-1 helicase-primase holoenzyme
[UL52(D628A)]; lanes3 and 8,
HSV-1 helicase-primase holoenzyme [UL52(D630A)]; lanes4 and 9, HSV-1 helicase-primase holoenzyme
[UL52(N624G)]. Lanes5 and 10 contained no added helicase-primase. Assays analyzed in lanes6-10 included the HSV-1 DNA polymerase holoenzyme
and the four dNTPs. Origin, the electrophoretic origin of the
gel; 20nt, 16nt, 12nt, and 10nt, the positions of
reference oligonucleotides electrophoresed in parallel
lanes.
or
Asp
) dramatically reduced the in vitro DNA
priming activity of the HSV-1 helicase-primase holoenzyme. From the
gel-based assay that directly examined RNA primer synthesis by the
HSV-1 helicase-primases, the reduction in primase activity is estimated
to be
100-fold. The HSV-1 helicase-primase [UL52(N624G)]
functioned similarly to the wild-type enzyme. This implies that despite
a primary amino acid sequence proximity, the conserved Asn
does not participate directly in mediating RNA primer synthesis.
. This NTP-binding site is unrelated to the NTPase
site on the HSV-1 helicase-primase holoenzyme. Alteration of the UL5
NTP-binding motif results in enzyme devoid of NTPase and helicase
activities with enhanced primase activity.
(
)
At
this time, it is not known which additional structures on the UL52
protein mediate other functions involved in RNA primer synthesis. Other
studied DNA polymerases and primases have been found to contain all the
structural domains necessary for nucleotide primer elongation on a
single polypeptide chain. It is likely this may be the case with the
UL52 subunit of the HSV-1 helicase-primase.
(
)
This structure may
contribute to a mechanism that coordinates Okazaki strand RNA priming
with duplex DNA unwinding at the advancing herpes replication fork.
Ongoing studies are focused on delineating additional functional
domains within the UL52 protein of the HSV-1 helicase-primase.
Table:
Primase activity of wild-type and altered HSV-1
helicase-primase holoenzymes
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.