(Received for publication, December 20, 1994)
From the
A number of studies have demonstrated that the herpes simplex virus type 1 (HSV-1) UL9 protein, which is a homodimer in solution, binds to two high affinity binding sites in each origin of replication. Interaction between the proteins bound at the two sites leads to the formation of a complex nucleoprotein structure. The simplest models for this binding interaction predict two possible binding stoichiometries: 1) one UL9 dimer is bound at each site; or 2) one UL9 monomer is bound at each site so that one UL9 dimer occupies both sites. Two recent papers have addressed this issue by using indirect methods to measure the binding stoichiometry. Martin et al. (Martin, D. W., Muñoz, R. M., Oliver, D., Subler, M. A., and Deb, S.(1994) Virology 198, 71-80) reported that a monomer of [Medline] UL9 binds to a single high affinity site, and Stabell and Olivo (Stabell, E. C., and Olivo, P. D.(1993) Nucleic Acids Res. 21, 5203-5211) concluded that a dimer of UL9 binds to a single high affinity site. We have directly measured the stoichiometry of binding of the carboxyl-terminal DNA binding domain of UL9 (t-UL9) to the origin of replication using a double-label gel shift assay. Using a short synthetic double-stranded oligonucleotide containing a single UL9 binding site, one protein-DNA complex was detected in the gel shift assay, and the molar ratio of UL9 DNA binding domains to DNA binding sites in this complex was determined to be 2.0 ± 0.1 (n = 13). Using the minimal origin sequence excised from plasmid DNA, two protein-DNA complexes were detected. The binding stoichiometry of the faster migrating complex was 1.8 ± 0.1 (n = 15), and the stoichiometry of the more slowly migrating band was 3.7 ± 0.4 (n = 15). The simplest explanation for these data is that UL9 binds to the origin of replication as a homodimer with one dimer bound at both high affinity sites.
The herpes simplex virus (HSV) ()genome contains
three cis-acting regions that function as origins of
replication: two identical copies of a sequence called ori
and one copy of a very closely related sequence called
ori
(3) . Although the mechanism by which DNA
replication is initiated at these sequences is not yet known, there is
considerable information regarding both the elements of the origin
sequence that are important for replication and the proteins with which
the origin interacts. Ori
contains at least five functional
domains: two high affinity binding sites (called site I and site II or
box I and box II) for UL9, a virally encoded DNA-binding protein that
is known to be essential for viral DNA
replication(3, 4, 5, 6, 7, 8, 9, 10) ;
an A/T-rich region; a sequence homologous to site I but with much lower
affinity for UL9, called site III (or box III); and a binding site for
an uncharacterized cellular protein(s) called OF-1(11) . Sites
I and II are located on the arms of a 46-base pair palindrome and
separated by the A/T-rich region. Nuclease protection, chemical
modification, and saturation mutagenesis studies have shown that the
high affinity recognition sequence for UL9 (site I) is contained in the
10-base pair sequence
5`-CGTTCGCACT(8, 9, 12) . Site II differs
from this sequence at two positions, resulting in a reduced binding
affinity for UL9 to about one-fifth that of site I (5, 8, 9) . Results of genetic studies have
suggested that the binding of UL9 to the origin is important for viral
DNA replication: mutations in ori
that decrease the ability
of UL9 to bind to site I or site II significantly decrease the
efficiency of replication of the ori-containing plasmid in transient
replication assays(13, 14) .
UL9 is comprised of
951 amino acids with a predicted molecular mass of 94 kDa and contains
at least two functional domains: the carboxyl-terminal domain of 317
amino acids mediates sequence-specific DNA
binding(1, 2, 7, 9, 15, 16, 17) ,
and the amino-terminal two-thirds of the protein mediates a
DNA-dependent helicase activity, allowing UL9 to unwind partially
duplex DNA of nonspecific
sequence(18, 19, 20, 21) . In
addition, this domain also appears to mediate dimerization and
protein-protein interactions between UL9
molecules(22, 23) . UL9 has not been shown to unwind
fully duplex DNA or to unwind preferentially origin-containing
sequences(19, 24) . Nevertheless, by analogy with
better characterized viral initiator proteins such as the simian virus
40 T antigen or the O protein (reviewed in 25), UL9 may initiate
HSV DNA replication by binding to the origin and unwinding a local
region of DNA to allow or direct the assembly of the HSV DNA
replication machinery.
UL9 is a homodimer in solution (18, 19) and binds to ori in a cooperative
manner at sites I and II(8, 22, 23) . The
simplest models for the binding of UL9 to the origin, therefore, entail
the binding of either one UL9 dimer to ori
, with a monomer
unit bound each at site I and site II, or two UL9 dimers bound to
ori
, one each to site I and site II. It is likely that a
complete understanding of the early events of viral DNA replication
will depend on an accurate determination of the binding stoichiometry
between UL9 and the origin. Recently, two groups have used the
carboxyl-terminal 317-amino acid DNA binding domain of UL9 in a gel
shift assay to measure the binding stoichiometry between UL9 and the
origin. The two groups, however, obtained opposing results: Martin et al.(1) reported that a monomer of UL9 binds to a
single high affinity UL9 binding site; in contrast, Stabell and Olivo (2) concluded that a dimer of UL9 binds to a single high
affinity site. These two results cannot be reconciled easily. The
conclusions drawn from these gel shift assays, however, were
inferential; both indirectly measured the binding stoichiometry of UL9
and ori
. We have, therefore, directly measured the
stoichiometry of binding between the carboxyl-terminal DNA binding
domain of UL9 and the origin of replication. In this paper, we show
that two monomer DNA binding domains of UL9 bind to a single high
affinity UL9 binding site. Furthermore, in the context of the intact
ori
, four monomer UL9 binding sites are bound: two each to
site I and site II.
The DNA binding domain of UL9 (t-UL9) was used to determine
the binding stoichiometry for UL9 and ori by using a
double-label gel shift
assay(
)(31, 32, 33, 34, 35, 36, 37) .
The truncated protein rather than the full-length protein was used for
two reasons. First, t-UL9 causes a much simpler pattern in gel shift
experiments than UL9, probably because it does not have a tendency to
aggregate. Second, t-UL9 has been reported to be a monomer in solution,
a fact that simplifies the interpretation of binding stoichiometry
measurements. A recombinant baculovirus was constructed which expresses
a polypeptide comprising amino acids 534-851 of UL9, with an
oligo-histidine affinity tag placed at the amino terminus to facilitate
the isolation of chemically homogeneous protein of high specific
activity. The protein was labeled in vivo with
[
H]leucine and was purified to chemical and
radiochemical homogeneity in a two-step process from the infected
nuclear extract using nickel-agarose affinity chromatography and gel
permeation chromatography. The silver-stained polyacrylamide gel of the
peak fractions from the gel permeation column and the corresponding
fluorogram of the silver-stained gel are shown in Fig. 1. The
minor band migrating at a slightly lower molecular weight from t-UL9
was seen in varying proportions to the major band in different t-UL9
preparations and is likely to be a proteolytic product of t-UL9. The
hydrodynamic properties(22) , DNase I footprint
pattern(2, 22) , and gel shift
pattern(1, 2, 7, 15, 16, 23) of the
H-labeled His-tagged t-UL9 were the
same as those described previously for similar untagged molecules. The
molar extinction coefficient of His-tagged t-UL9 was calculated to be
37,830 M
cm
, using the
method of Gill and von Hippel(28) . The absorbance at 280 nm of
t-UL9 denatured with 6 M guanidinium HCl, however, was 10%
lower than the absorbance of the native protein. The molar extinction
coefficient of the
H-labeled t-UL9 was therefore adjusted
to 34,047 M
cm
, and the
specific activity was calculated to be 27,978 dpm/pmol.
Figure 1:
Silver stain and fluorogram of
polyacrylamide gel containing the peak fractions of the final step of
purification of H-labeled t-UL9. One µl of each of the
load and peak fractions from the Smart/Superose 12 column was run on a
4-20% polyacrylamide gel(27) . The gel was stained with
silver, soaked in Amplify, dried, and subjected to autoradiography. The
fraction numbers are listed across the top of the gel. For
column calibration, Bio-Rad molecular mass standards
-globulin
(160 kDa), ovalbumin (44 kDa), and ribonuclease A (17 kDa) eluted in
fractions 15, 22, and 28, respectively.
Varying
amounts of H-labeled t-UL9 were mixed with a fixed amount
of
P-labeled 24-base pair double-stranded oligonucleotide
containing site I. The protein-DNA complexes were separated from the
unbound DNA by nondenaturing polyacrylamide gel electrophoresis. As
seen in Fig. 2, a single lower mobility band was seen at all
t-UL9 concentrations. The lower mobility complex in each lane was
excised from the gel, dissolved, and the amount of
H and
P radioactivity determined by liquid scintillation
counting. The results from this experiment are shown (Fig. 2).
The labeled bands contained 1
10
to 1
10
dpm of
H and
P radioactivity,
with
H dpm in approximately 3-fold excess over
P dpm; both amounts are in a range where counting errors
are negligible. In a separate experiment similar to that shown in Fig. 2, one lane of the gel was cut into 0.5-cm slices, and the
radioactivity in each slice was determined. As depicted graphically in Fig. 3, the
H-labeled protein was associated
predominately with the
P-labeled DNA and was not
distributed throughout the gel. The ratio of t-UL9 monomers to UL9
binding sites was determined to be 2.0 ± 0.1 (n = 13).
Figure 2:
Autoradiogram of a double-label gel shift
experiment using H-labeled t-UL9 and
P-labeled
site I oligonucleotide.
H-Labeled t-UL9 (27,978 dpm/pmol)
was titrated into reaction mixtures containing 5 pmol of 32P-labeled
site I oligonucleotide (19,111 dpm/pmol): lane 1, no added
protein; lanes 2-6, 3, 6, 9, 12, and 18 pmol of
H-labeled t-UL9, respectively. The protein-DNA complexes
were separated from the unbound DNA fragments on a 5% nondenaturing
polyacrylamide gel. The gel was exposed to x-ray film at room
temperature while wet, the lower mobility bands (at the position
indicated by the arrow) were cut from the gel, and the slices
were dissolved and counted. The
H dpm and
P
dpm of each excised gel slice are listed below the corresponding lower
mobility band.
Figure 3:
Plot of radioactivity versus distance migrated. A representative lane from an experiment
similar to that shown in Fig. 2was sectioned into 0.5
1.0-cm slices from the well to the bottom of the gel. The slices were
dissolved, counted, and the data plotted. The slower mobility peak in
the plot corresponds to the position on the autoradiogram of the gel of
the low mobility complex. The faster mobility peak in the plot
corresponds to the position on the autoradiogram of the gel of the
faster mobility complex.
To control for possible errors that might have
been introduced by the use of a synthetic oligonucleotide, a DNA
substrate prepared by a different process was also used. HSV-2
ori was excised from highly purified plasmid DNA,
endlabeled with
P, and the labeled DNA fragment was
purified by agarose gel electrophoresis. The gel shift assay was
performed as described above with the synthetic oligonucleotide. The
addition of
H-labeled t-UL9 to reactions containing this
DNA fragment resulted in the appearance of two lower mobility
complexes, with the faster mobility of the two predominating at low
t-UL9 concentrations and the slower mobility complex predominating at
higher concentrations of t-UL9 (Fig. 4). DNase I footprint
analysis of the DNA in the two complexes revealed protection over sites
I and II only, with more complete protection of both sites I and II in
the lower mobility complex (data not shown). The ratio of monomer t-UL9
molecules to UL9 binding sites was 1.8 ± 0.1 (n = 15) for the higher mobility complex and 3.7 ± 0.4 (n = 15) for the lower mobility complex. These data are
in close agreement with those obtained with the synthetic
oligonucleotide that contained a single UL9 binding site. Thus, two UL9
binding domains are necessary and sufficient for binding to a single
UL9 binding site. These findings, therefore, confirm the previously
published results of Stabell and Olivo(2) . In addition, these
results show that four UL9 binding domains are bound to
ori
. Taken together, these data are consistent with a model (Fig. 5) in which two UL9 dimers are bound to ori
,
one each at site I and site II.
Figure 4:
Autoradiogram of a double-label gel shift
experiment using H-labeled t-UL9 and
P-labeled
ori
.
H-Labeled t-UL9 was titrated into reaction
mixtures containing 0.6 pmol of 32P-labeled ori
: lane
1, no added protein; lanes 2-6, 0.8, 1.7, 3.3, and
6.6 pmol of
H-labeled t-UL9, respectively. The protein-DNA
complexes were separated from the unbound DNA fragments on a 5%
nondenaturing polyacrylamide gel. The gel was exposed to x-ray film at
room temperature while wet, the bands were cut from the gel, and the
slices were dissolved and counted. The ratio (mean of all experiments, n = 15) of
H-labeled t-UL9 molecules to
P-labeled ori
molecules is reported next to
the position of the corresponding band.
Figure 5:
Model diagram depicting the binding of
ori by: A, t-UL9; and B, UL9. A/T is the A/T-rich region between the two UL9 binding sites in
ori
. The bipartite shaded regions of sites I and
II depict the overlapping inverted binding half-sites. The small, lightly shaded area between monomer UL9
molecules of the UL9 homodimer represents the monomer-monomer
interaction between UL9 molecules of the homodimer molecule. The larger shaded regions between UL9 homodimers bound to
ori
represent the interaction between UL9 homodimers as
suggested by the cooperative binding of UL9 to
ori
.
It is clear that these conclusions
are critically dependent on the accuracy of the measurements. Since the
standard error derived from the average of 15 independent measurements
was between 1 and 3%, it seems unlikely that the conclusions are
affected by random experimental error. Possible sources of systematic
error, and the potential magnitude of such errors, are, however, worth
noting. In addition to the quantity of H and
P
radioactivity present in various samples, the only other measured
values in the described experiments were the chemical amounts of DNA
and protein. In both cases, these quantities were determined
spectrophotometrically, and therefore the conclusions depend on the
accuracy of the extinction coefficients used. The extinction
coefficient of double-stranded DNA at 260 nm is known precisely and is
essentially independent of the sequence of the DNA(3) . In
addition, as an internal control in our determinations of the binding
stoichiometry, two different DNA molecules isolated from independent
sources were used, and essentially the same result was obtained with
each. The specific activity of the
P-labeled DNA,
therefore, is unlikely to be a source of significant error in the
calculation of the binding stoichiometry.
Precise determination of the extinction coefficient of t-UL9 is somewhat more problematic. In contrast to DNA, proteins do not have a uniform extinction coefficient at 280 nm. The method of Gill and von Hippel (28) was used to derive the extinction coefficient for t-UL9. This method is based on the demonstration by Edelhoch (40) that only tryptophan, tyrosine, and cysteine residues contribute significantly to the measured optical density of a denatured protein at 280 nm. The extinction coefficient of the protein of interest is calculated by multiplying the number of residues of each of the three absorbing amino acids by the molar extinction coefficients of model peptides containing these residues. This method for calculating molar extinction coefficients was calibrated by calculating the extinction coefficients for 18 globular proteins whose extinction coefficients were accurately known(28) . The mean deviation between the experimental and the calculated values was +3.8% ± 6%, with no deviation greater than +14.9% (for human serum albumin). Since the method was calibrated using native proteins, the largest potential error in this calculation is likely to be the effect of tertiary structure of the native protein on the absorbance of the individual absorbing amino acids. This effect was measured by comparing the absorbance at 280 nm of native and denatured t-UL9, as described under ``Experimental Procedures.'' A small difference (10%) between the absorbance of the native and denatured proteins was observed, and the extinction coefficient of t-UL9 was adjusted appropriately. On the basis of this small difference in absorbance between the native and denatured protein and on the basis of the prior results of Gill and von Hippel, the estimated error in the calculated value for the extinction coefficient of t-UL9 is less than 15% and is likely to be much lower.
Recently,
two other laboratories have used gel shift assays to measure the
binding stoichiometry between t-UL9 and the origin; the results of
these two studies were not in agreement. Stabell and Olivo (2) titrated Fab fragment directed at the carboxyl terminus of
UL9 into a gel shift mixture containing t-UL9 bound to a single UL9
binding site and detected two bands of lower electrophoretic mobility,
suggesting that two t-UL9 molecules bound the site. Martin et al.(1) used in vitro transcription/translation to
obtain t-UL9 and a slightly truncated t-UL9 molecule. In gel shift
experiments in which both of these forms of t-UL9 were present, the
investigators observed only two complexes, the mobilities of which
corresponded to the mobilities of the complexes observed with each form
of t-UL9 alone. This result suggested that no heterodimers between the
larger and smaller polypeptides were formed during cotranslation and
therefore that t-UL9 bound to a single binding site as a monomer. The
disparate conclusions from these sets of experiments are difficult to
reconcile; both, however, required significant assumptions about the
gel shift banding pattern without knowledge of the composition of the
bands. The experiments presented in this paper have significant
advantages over the indirect approaches described above. Foremost, no
assumptions are required as to the interpretation of the banding
pattern in the gel shift experiments, as the composition of each
complex was determined directly. In addition, the t-UL9 used in the
double-label gel shift experiments was highly purified and well
characterized, and secondary heterologous protein binding was not
required for the stoichiometric determinations. The conclusions,
therefore, are based on direct measurements of physical constants and
require only that the labeled protein and DNA were chemically pure and
that the extinction coefficient for t-UL9 was determined accurately.
The results, then, confirm those of Stabell and Olivo(2) , that
t-UL9 binds to a single high affinity UL9 binding site as a dimer, and
they extend the findings to demonstrate that two t-UL9 dimers bound to
ori.
Taken together, the simplest interpretation of
these data is that one full-length UL9 dimer binds to a single UL9
binding site and that two full-length UL9 dimers bind to ori (Fig. 5). The underlying assumption of this model is that
the results obtained with t-UL9 can be extrapolated directly to what
occurs with the full-length protein. Other more complicated models,
however, are consistent with the available data. For example, it is
possible that full-length UL9 monomers comprising a UL9 dimer are
oriented so that the two monomeric units cannot both bind to the same
DNA binding site. According to this model, a single binding site would
be occupied by two UL9 dimers, with two unoccupied monomeric units
available to bind to another site. To address this question, the
double-label gel shift experiment was employed using
-
H-labeled full-length UL9. As has been reported
previously(23, 38) , UL9 and the site I
oligonucleotide formed a large number of complexes that were resolvable
by gel electrophoresis. Moreover, the complexes did not differ in the
protein:DNA ratio by an integral number of UL9 molecules, nor was there
a monotonic increase or decrease in the protein:DNA ratio of the
complexes as a function of electrophoretic mobility (data not shown).
We currently have no simple interpretation of these data, although we
assume that the large number of protein-DNA complexes formed with
full-length UL9 is related to its tendency to aggregate. In any case,
these data do not distinguish between the model presented in Fig. 5and alternative models.
The binding of two t-UL9
monomers to a specific binding site is highly cooperative, since no
intermediate t-UL9-site I complex containing a single monomer of t-UL9
was detected (see Fig. 2and Fig. 4). The simplest
explanation for this high degree of cooperativity is that t-UL9
monomers interact with each other. As noted above, t-UL9 is a monomer
in solution, at least at concentrations less than 1
10
M. Higher order association between
t-UL9 molecules in concentrated solutions has been observed, however, (
)(
)and experiments designed to confirm this
finding using highly purified protein are in progress. There are also
other possible explanations for the highly cooperative nature of t-UL9
binding to site I. For example, it has been reported that UL9 binding
to the origin distorts the DNA near its binding site(39) . It
is possible that the change in free energy associated with this DNA
conformational change contributes to cooperativity. Alternatively, the
t-UL9 may undergo conformational changes upon binding to DNA that
increase the magnitude of protein-protein interactions. A rigorous
analysis of the energetics of UL9-DNA interactions will be required to
distinguish among these possibilities.
The finding that two UL9 DNA
binding domains bind to a single binding site predicts that the DNA
sequence of the binding site should contain a 2-fold axis of symmetry.
The consensus 10-base pair UL9 recognition site 5`-CGTTCGCACT, however,
does not contain a simple 2-fold axis of symmetry. Koff and Tegtmeyer (12) have suggested that similar to the SV40 T antigen
recognition sequence, site I is composed of inverted overlapping
repeats that could be the binding half-sites for UL9. A more recent
mutational analysis of ori by Hazuda et al.(9) has provided data consistent with this hypothesis.
Although it would be without precedent, it is also possible that the
UL9 homodimer binds asymmetrically to DNA. Further, more detailed
investigation of the binding of UL9 to its recognition sequence will be
required to determine the exact nature of this binding interaction.