From the Department of Molecular and Cell Biology,
University of California, Berkeley, California 94720 and the
¶ Department of Cell Biology and Neuroanatomy, University of
Minnesota Medical School, Minneapolis, Minnesota 55455
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ABSTRACT |
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DNA replication initiator proteins bind site
specifically to origin sites and in most cases participate in the early
steps of unwinding the duplex. The papillomavirus preinitiation complex that assembles on the origin of replication is composed of proteins E1
and the activator protein E2. E2 is an ancillary factor that increases
the affinity of E1 for the ori site through cooperative binding. Here
we show that duplex DNA affects E1 (in the absence of E2) to assemble
into an active hexameric structure. As a 10-base oligonucleotide can
also induce this oligomerization, it seems likely that DNA binding
allosterically induces a conformation that enhances hexamers. E1
assembles as a bi-lobed, presumably double hexameric structure on
duplex DNA and can initiate bi-directional unwinding from an ori site.
The DNA takes an apparent straight path through the double hexamers.
Image analysis of E1 hexameric rings shows that the structures are
heterogeneous and have either a 6- or 3-fold symmetry. The rings are
about 40-50 Å thick and 125 Å in diameter. The density of the
central cavity appears to be a variable and we speculate that a plugged
center may represent a conformational flexibility of a subdomain of the
monomer, to date unreported for other hexameric helicases.
The synthesis of duplex DNA is a complex enzymatic process that
requires the coordination of large numbers of proteins. The mechanisms
are elaborate in part because the enzymes that use the complementary
template strand as a guide for nucleotide incorporation catalyze this
synthesis only in the 5' to 3' direction. Given the antiparallel nature
of the duplex this usually requires that two synthetic enzymes move in
opposite polarities on the two strands. Synthesis of the so-called
lagging strand is discontinuous and requires the cyclical association
of the enzyme, while synthesis of the other strand is continuous.
Nevertheless, in many prokaryote replication systems it is clear that
coordination of these enzymes is achieved and maintained by a dimeric
polymerase that creates a looped DNA structure in the lagging strand.
This loop is mediated by multiple protein-protein interactions across
the growing fork (Ref. 1, and references therein). Helicases are
enzymes that can catalyze the unwinding of the template strands ahead
of the fork, thus allowing for new complementary strand DNA synthesis. They were initially discovered as ancillary factors required for synthesis, but recently this view of the helicase activity has been
characterized as "naive" or at least incomplete (2). Compelling evidence has been presented demonstrating that the helicase is an
integral member of a large protein complex that serves as a molecular
motor or pump for the replication apparatus empowering the polymerase
and increasing the rate of DNA polymerase synthesis (3). The
Escherichia coli dnaB helicase also plays a critical function in establishing the asymmetry at the growing fork. The helicase tracks on the lagging strand template but through interactions it holds the leading strand DNA polymerase while allowing for recycling
of the other DNA polymerase (4). How helicases actually convert the
binding and hydrolysis of ATP into mechanical energy resulting in DNA
unwinding and can concomitantly achieve relative movement along the DNA
is presently under intense investigation (5, 6). While many issues
remain unresolved, it seems as if the well studied replication
helicases of E. coli (and its phage encoded ones) engage DNA
by encircling at least one of the DNA strands that have been prepared
for this loading by other replication proteins (7-10). Thus, for
example, the dnaB helicase is loaded onto DNA in complex with dnaC to a
duplex structure at oriC already melted by the dnaA protein (11).
In eukaryotes, despite the ubiquitous presence of many DNA helicases
(12), little is known about the relationship between such enzymes and
the replication complex. However, the importance of such proteins in
eukaryotic DNA replication is highlighted by the fact that many DNA
viruses that replicate in the nucleus encode a helicase. One type of
such viral helicase can initiate unwinding from within duplex
structures not prepared for activity by prior melting. These helicases
encoded by the herpes simplex virus, papillomaviruses or the SV40, and
polyoma viruses can serve as DNA initiators by first recognizing small
repeat motifs within the origin of replication. Thus, a particularly
challenging structural problem exists in determining how these proteins
convert from a site-specific DNA binding mode to a helicase. For the
SV40 T antigen, monomers bind to pentameric base pair repeats utilizing specific nucleotide base information. After double hexamer formation on
the DNA and ATP binding DNA-protein contacts shift toward
sugar-phosphate interactions (13). A complex series of steps must
therefore occur to change both the oligomeric state of the protein and
the nature of its contacts with DNA. Presumably, both the DNA and ATP
could be allosteric effectors of this change, but in the case of T
antigen ATP is sufficient for hexamer formation. Similarly for the
HSV-1 origin binding protein UL9 a pair of dimers interact with each
other and bend the ori region as duplex DNA-binding proteins. In the
presence of ATP the complex becomes an active unwinding enzyme that can
extrude catenated single-stranded loops (14).
The papillomaviruses provide a unique system for analyzing this
assembly and transition process. The bovine papilloma virus type 1 (BPV-1)1 encodes a 68-kDa
phosphoprotein (E1) that binds site specifically to two sets of short
repeats organized as inverted repeats at the viral origin of
replication (15, 16). The protein is also an ATPase (17) and a helicase
able to initiate unwinding from within a duplex DNA circle (18, 19).
However, unlike UL9 or the SV40 T antigen, E1 requires in
vivo an ancillary viral factor in order to be targeted to the
viral origin of replication (20, 21). The E1 protein in a cell-free DNA
replication system can direct origin-specific DNA replication; however,
this activity is greatly stimulated by E2 and at limiting dilutions of
E1 the in vitro DNA replication becomes absolutely dependent
upon E2 (22). This dependence upon E2 reflects the cooperative
interaction between E1 and E2 in binding to viral DNA (22-25). The
E2·E1·DNA complex initially assembled at the origin consists of two
or four monomers of E1 spanning the inverted repeats and an E2 dimer
occupying an adjacent binding site (15). Interestingly, the E2 dimer
must leave this complex in an ATP-dependent reaction before
higher-order complexes of E1 assemble (26, 27). Therefore, it seems as if E2 might stabilize the site-specific DNA binding conformation of E1
and either mask or allosterically block the helicase transition. In vivo, this chaperone process seems likely to have evolved
to increase the likelihood of origin occupancy and perhaps to allow for
the coordination of other activities of E1 with the transcriptional and
segregation functions of E2 (28, 29). In any case understanding the
differences between the E1 assembled in the preinitiation E1·E2
complex and as an active helicase on the DNA should provide insights
into the transitions that must occur for all of the viral initiator
proteins discussed above.
E1 is believed to be structurally related to the SV40/polyoma
virus-encoded large T antigens. Both helicases track on the leading
strand template (23), and the overall organization of the open reading
frames are similar. For instance, the nuclear localization domain is
proximal to the site-specific DNA-binding domain and the conserved
"Walker" A and B boxes of the ATP-binding domains are equivalently
spaced and are about 200 amino acids displaced from the DNA-binding
domain (30). Moreover, mutations of these conserved motifs affect
activity in expected ways (17, 19, 31, 32). However, many of these
alignments can now be made for other members of the SF3 family of DNA
helicases that do not have analogous activities such as those from
parvoviruses and the human herpes 6 virus (33). It is therefore
important to analyze how E1 as an active unwinding enzyme actually
engages DNA. In this report we use both biochemical and electron
microscopy techniques to establish that E1 initiates unwinding from the
origin site and unwinds DNA bidirectionally; moreover a bi-lobed double hexameric complex similar to the images obtained for T antigen was
observed at the start site. In these complexes the DNA is likely to
take a straight path through the double rings. Biochemical and electron
microscopic analysis showed that, as anticipated, unwinding activity
correlates with the formation of hexamers. Image analysis of the
hexameric structures showed that the molecules do form toroidal rings
with a central hole; these molecules possess either 3- or 6-fold
symmetries. Surprisingly, a significant fraction of the hexamers show
density in the central hole. Such "filled" centers have not been
observed for other hexameric helicases but we speculate that the
protein has several conformational states and that conformational
flexibility may indeed be a general feature of this family.
Plasmid Construction and DNA Substrates--
pKSO has been
described previously (22). pSS3, pSS3-LI5C, and pSS3- Protein Purification--
The BPV-1 E1 protein was purified from
Sf9 cells infected with a recombinant baculovirus expression
vector by immunoaffinity chromatography as described by Yang et
al. (22). The E1 protein purified from E. coli began
with transforming XA90 cells with the pGEX-2TK-E1 expression vector and
proceeded according to methods described by Sedman et al.
(25) as modified by C. Sanders.2 In brief, extracts
from isopropyl-thio- Unwinding Assays--
The unwinding reactions using either
covalently closed circular DNA or duplex DNA fragment substrates were
performed as described previously (19).
Electron Microscopy and Measurement of DNA
Regions--
Unwinding reactions using substrates indicated in the
text were incubated at 32° C for 1 h. Micrographs of linearized
DNA were prepared by adding 6 units of the indicated restriction enzyme and incubating for an additional 20 min. Reaction products were fixed
by the addition of glutaraldehyde to 0.6% and purified by filtration
through a 0.5-ml Bio-Gel A5-M column (Bio-Rad) and applied to
glow-discharged carbon grids coated with 2 mM spermidine. The grids were then rotary shadowed with tungsten. Photographs were
taken at × 30,000 with a JEOL 1200 EX electron microscope at an
acceleration voltage of 80 kV (61). Micrographs of E1 complexes bound
to a BPV-1 origin containing DNA fragment for linear compaction studies
were prepared using the same method described above. Measurement of
duplex regions was performed by projecting photographic negatives onto
a Numonics digitizing tablet.
Image Analysis--
The E1 protein (3 mM
concentration, in 25 mM KPO4, 60 mM
NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5) was
incubated with 18 mM of a 60-mer oligonucleotide for 30 min
at 30° C, and then applied to glow-discharged grids and stained with
2% (w/v) uranyl acetate. Electron micrographs were recorded under
minimal dose conditions (with no prior exposure to the high
magnification electron beam prior to recording) at × 30,000 magnification, using a JEOL 1200 EXII microscope. Negatives were
scanned with a Leaf 45 microdensitometer, with a sampling interval of 4 Å/pixel. Images of rings were masked into 44 × 44 pixel arrays
(corresponding to 176 × 176 Å), band-pass filtered (between
1/160 and 1/12 Å Radiolabeling of E1--
The 7 amino acid
NH2-terminal tag on the E1 purified from overexertion in
E. coli contains a recognition sequence as well as a serine
that can be specifically phosphorylated by bovine heart muscle kinase.
A typical 30-µl labeling reaction contains 2.5 µg of E1 (1.3 µM final), 10 units of bovine heart muscle kinase (Sigma
P-2645), and 2 µl of [ Electrophoresis of E1 Complexes--
Native gel electrophoresis
of E1 was performed as indicated in the text. For analysis of
cross-linked E1 complexes, the E1 sample was boiled for 5 min in
Laemmli SDS loading buffer (pH 8.8) + 130 mM
Glycerol Gradient Sedimentation--
E1 purified from Sf9
cells infected with a recombinant baculovirus was centrifuged in a
15-35% glycerol gradient with 0.1 KCl-HEMG + 0.01% Nonidet P-40
(62). For E1 purified from overexpression in E. coli, the
protein was centrifuged as above with the following modification: a
gradient of 15-37% glycerol in 1 M NaCl, 20 mM HEPES (pH 7.5), 5 mM EDTA, and 0.01%
Nonidet P-40 was used.
E1 Unwinds DNA Bidirectionally from the ori Site--
BPV-1
replicates bidirectionally in vivo and in cell-free extracts
(22, 34, 35). With purified replication components wherein E1 provided
the only helicase activity (36), fully replicated circles were
obtained. Furthermore, acting on covalent closed circles, E1 is capable
of producing a highly unwound DNA (form U) consistent with complete
denaturation of the circles (19, 23). It was therefore anticipated that
E1 as a replicative helicase must be capable of processive unwinding
that spreads bidirectionally from the BPV-1 origin region. Electron
microscopy and an in vitro unwinding assay were employed to
map both the location and extent of unwinding of BPV-1 genomic DNA by
E1. Covalently closed circular DNA was relaxed with calf thymus
topoisomerase I and then incubated with purified E1, ATP, topoisomerase
I, and E. coli SSB protein. For this analysis, we used the
plasmid pSS3 that contains an intact BPV-1 genome cloned into pUC18.
The samples were fixed with glutaraldehyde, purified by gel filtration,
and linearized by restriction endonuclease hydrolysis at a unique site.
Representative images are shown in Fig.
1, A-D. The lengths of the
unwound regions of DNA and total contour lengths were measured to
determine both the size and position of the unwound regions with
respect to the entire length. A compilation of data is presented in
Fig. 1E. As each of the molecules could be aligned in either
of two directions (and one such orientation chosen for each to make the
alignment) it was necessary to repeat this analysis with a different
unique single cutter. This separate set increases the statistical
significance of the conclusions. Such data obtained with either
AflII or SacI defining the ends (Fig.
1E) show that unwinding initiates at the ori site and that denaturation spreads bidirectionally from that position.
These results are compatible with those presented previously by Seo
et al. (18), who showed that E1-dependent
formation of form U was dependent upon the integrity of ori. However,
our earlier results (19) on form U production showed little dependence upon origin sequences, a result that might predict scattered bubble positions. With different preparations of the E1 protein we performed in vitro unwinding assays with various mutant templates.
Mutations were engineered into the E1-binding site to determine whether the E1 DNA-binding site contributed quantitatively to the level of form
U DNA produced. Two mutants were derived from the plasmid pSS3; LI5C
contains a 5-base pair linker insertion between the inverted repeat,
and for The E1 Protein Forms Hexamers and Oligomerization Correlates with
Helicase Activity--
To determine the oligomeric states of E1 we
analyzed the sedimentation profiles of the baculovirus-purified E1
incubated in the presence and absence of ATP. Fractions from a glycerol
gradient were collected, and the positions of the E1 protein determined by SDS-PAGE and Western blotting using polyclonal E1 antisera. The
protein profiles of the gradients (Fig.
3) indicate that E1 purified by single
step affinity chromatography sediments in a heterogeneous manner. In
addition, it is clear from this analysis that ATP is not sufficient for
the oligomerization of E1 monomers. The molecular mass markers suggest
that the slowest migrating peak corresponds to monomeric E1 (68 kDa)
and the second peak a hexameric form (408 kDa). Fractions at the bottom
of the tube likely correspond to aggregates. Glycerol gradient
fractions containing the putative hexamer fraction and monomer
fractions were analyzed by electron microscopy. The images of the
putative hexamer peak showed a typical 6-membered toroidal structure
(Fig. 3) bearing striking similarity to the published micrographs of
hexameric helicases (8, 10, 37-39). The monomeric peak showed no such structures (data not shown).
Protein preparations from baculovirus vectors showed a mixture of
forms, and to study a more homogeneous population and to investigate
the relationship between the monomer and hexamer forms in more detail,
we purified E1 from E. coli cells using the methods described by Sedman et al. (25). Indications from
spectrophotometric 280/260 absorption ratios for baculovirus E1
preparations were that the yields of hexameric peaks and aggregated
material correlated with trace nucleic acid contaminations. We
therefore explored the notion that DNA binding might be a factor in
oligomerization. The E. coli E1 possesses an amino-terminal
sequence which can be phosphorylated to high specific activity in
vitro; such modification has no effect upon helicase activity or
other biochemical tests described below (data not shown). The purified
material sedimented as a homogeneous monomeric fraction (Fig.
4B). Moreover, this protein's
sedimentation behavior was not affected by ATP binding. The E. coli protein was found to be active in cell-free DNA replication and its activity was stimulated by E2 (data not shown).
The monomeric radiolabeled E1 was incubated with a 429-bp duplex DNA
fragment in the absence of ATP and Mg2+. Reactions were
fixed employing titrations of glutaraldehyde and subjected to
denaturing polyacrylamide gradient electrophoresis (Fig.
4A). A ladder of cross-linked phosphorylase or commercial prestained standards (Kaleidoscope, Bio-Rad) were used as gel standards. The results show that duplex DNA can promote oligomerization and the data confirm that hexameric forms of E1 predominate. In the
absence of DNA no multimerization was detected at any concentration of
cross-linking agent. These data were obtained both with nonspecific duplex and single-stranded DNA. Even very small oligonucleotides can
catalyze this oligomerization. In the presence of a 10-base single
strand oligomer (in the absence of ATP), E1 sediments as a hexamer in a
glycerol gradient (Fig. 4B). Analysis of the protein peaks
collected from these gradients (after glutaraldehyde fixation) by
denaturing acrylamide gel electrophoresis confirms that the high and
low molecular weight peaks represent hexameric and monomeric E1 forms
(Fig. 4C).
To explore the functional significance of this oligomerization we
sought to correlate E1 unwinding activity with its multimeric state. To
obtain such correlations we chose the duplex unwinding assay utilizing
a duplex restriction fragment that contains the BPV-1 ori sequence. In
previous experiments from our laboratory (19), unwinding in this assay
was dependent upon ori sequences and absolutely required an SSB. In two
parallel sets of reactions we either followed the oligomeric state of
E1 across a range of protein concentrations (3.75 to 480 nM) (Fig. 5A) or
the state of the duplex DNA (4.2 nM) over the same protein
titration (Fig. 5B). In these side-by-side experiments
reaction conditions were identical with the exception that SSB was not
present in the data obtained for Fig. 5A. As E1 assembles on
the DNA in the presence of ATP/Mg2+ and E. coli
SSB, the duplex is melted and converted to single strands. The ability
of E1 to act as a duplex unwinding enzyme is very cooperative (Fig.
5C) with respect to concentration and correlates with
oligomerization. Hexamers and high forms (perhaps double hexamers)
correlate with such activity. At 60 nM E1 some unwinding is
first detected and at this concentration hexamers and notably higher
forms are first detected.
DNA Takes an Apparent Linear Path Through a Bilobed E1
Complex--
From the data in Fig. 5 there is a suggestion that
complexes of higher order than hexamer may be the most efficient in
unwinding duplex DNA. For SV40 T antigen direct experiments indicate
that a double hexameric form of this helicase may provide the most effective enzymatic complex for such purposes (40, 41). To extend this
comparison we asked if a bilobed structure, taken as a measure of
double hexamer formation for T antigen (42, 43) could be observed for
E1. Purified E1 protein was incubated (in the absence of ATP) with a
242-base pair duplex DNA fragment containing a centrally located ori
sequence. Electron micrographs were recorded of the protein-DNA
complexes (Fig. 6A). The
characteristic "double doughnut" images were prevalent. The length
of the DNA fragments with and without bound protein was measured by
projecting electron micrograph negatives onto a numonics digitizing
tablet (Fig. 6B). We found that the DNA length distribution
does not change upon engaging E1. This absence of a linear compaction
of the protein-bound DNA indicates that the DNA likely takes on a linear path through the E1-protein complex. The data clearly rule out
any mode of binding which would require the DNA to wrap around the
helicase (Fig. 6C). Similar DNA compaction studies performed on the E. coli dnaB helicase (7) and T antigen (42, 43) have
been similarly interpreted; however, we would point out that the
standard deviation in length measurements (~9%) is very close to
what might be expected for more complex models wherein one strand
passes through one hexamer and out through the top of the same shell
(see line 2, Fig. 6C). To resolve this point higher resolution or other approaches to analyzing these structures is required.
Image Analysis of E1 Hexameric Ring--
To obtain a clearer
analysis of the ring structures, minimal dose electron microscopy and
image analysis were used to study the organization of the oligomeric
state of the E1 protein. Fig. 7 shows
electron micrographs where both top views of the rings (Fig.
7a, formed with an oligonucleotide) and side views of the rings stacked on double-stranded DNA (Fig. 7B) can be seen.
Images of 968 top views of the rings were averaged together, using a reference-free alignment procedure (44). The resulting average (not
shown) suggested a hexameric structure, but subsequent analysis indicated that the population of rings was non-homogeneous. First, a
sorting by rotational power (as done for DnaB protein in (39)) indicated that a subset of the rings had a significant 3-fold rotational power, consistent with the asymmetric unit in these rings
being a dimer. Trimers of dimers has previously been observed for the
hexameric rings formed by the DnaB protein (39, 45) and the RecA
protein (46). Fig. 8, a and
b, show the 6-fold symmetric averages (containing 678 rings), while Fig. 8, c and d, show the 3-fold
symmetric average (containing 100 rings). The rotational power spectra
for the 6- and 3-fold symmetric averages are shown in Fig. 8,
i and j, respectively. The main difference between the 6-fold symmetric rings and the 3-fold symmetric ones is
that there appears to be a modulation of the projected subunit density
in the 3-fold symmetric averages, such that there are alternating
"strong" and "weak" subunits. In addition, the outermost ends
of the subunit arms appear to move in toward the center for the three
weaker subunits in the 3-fold conformation. Both forms of the ring
appear to be about 125 Å in diameter.
Second, the density within the central channel appeared to be
continuously variable. The images contained in the 6- and 3-fold averages were then sorted based upon the strength of this central density. Fig. 8a shows an average of 400 6-fold symmetric
rings with a strong hole near the center, while Fig. 8b
shows an average of 278 6-fold symmetric rings with a "plug" of
density in the center. Similarly, Fig. 8c shows an average
of 56 3-fold symmetric rings with a strong hole near the center, and
Fig. 8d shows an average of 44 3-fold symmetric rings with a
plug in the center. There did not appear to be any correlation between
the strength of the 3- or 6-fold power and the strength of the central
density. Furthermore, both the relative strength of the 3-fold
rotational power and the relative strength of the central density
appeared to be continuously variable parameters. Thus, the groupings
that are shown in Fig. 8 represent arbitrary divisions. For example, averages could have been created in Fig. 8, b and
d, showing a stronger central density by using fewer images,
just as averages could have been generated in Fig. 8, c and
d, showing a slightly stronger 3-fold power by using fewer images.
Symmetrized versions of the averages in Fig. 8, a-d, are
shown in Fig. 8, e-h, respectively. One consequence of the
symmetrization, which eliminates noise, is that asymmetric features
disappear. However, this can also obscure real asymmetric features. The
central holes in Fig. 8, a and c, are displaced
from the central axis, while the symmetrization forces these holes to
lie on the central axis in Fig. 8, e and g. It is
likely that this displacement of the stain-filled hole from the central
axis is due to the binding of the 60-nucleotide oligomer, since this
mass might be expected to be bound within the central channel to only
one or two of the subunits, as shown for the T7 gp4 hexameric helicase
(10). Thus, the bound oligonucleotide would be filling some of the
central channel (indicated by arrows in Fig. 8, a and
c), leading to an asymmetric location of the stain-filled hole.
While the averages in Fig. 8 have a slight hand, the degree of
chirality is much less than that observed for DnaB (39, 45), T7 gp4
(10), or SV40 large T antigen (37). We therefore checked to see if the
lack of a strong hand arose from averaging together individual
projection images that were related by mirror symmetry. This would
occur if the rings were randomly oriented on the grid, as opposed to
predominantly one side adsorbing to the grid. We used the highly chiral
average of SV40 large T (37) as a reference to align all images, and
then did the alignment against the mirror image of the reference.
Images were ranked based upon the coefficient of correlation against
the reference, and averages were then created from those with the
strongest correlation using the reference-free alignment (44). The
results of this procedure suggested that there was no strong chirality
present, as no averages were generated with the opposite band to that
shown in Fig. 8. Thus, the rings appear to predominantly adsorb to the
grid by the same surface.
Conditions were found where "side" views of the E1 rings could be
obtained by binding them to double-stranded DNA molecules in the
presence of ATP (Fig. 7b). Fig. 7c shows an
average generated by aligning 217 side view images, each image
containing three rings. The spacing between adjacent rings is about 50 Å, and unless there is a large degree of interdigitation, this would
be the thickness of each ring. Since there does not appear to be a
large continuous density running between adjacent rings, a large degree of interdigitation appears unlikely. The spacings between the rings
were observed to be quite variable, and a number of different characteristic side views appeared to be present, as well. We therefore
sorted the images into subgroups using correspondence analysis (47) and
Fig. 7, d and e, show subaverages generated from
44 and 52 different rings, respectively. Since the alignment method
used to generate the averages in Fig. 7, d and e,
only examined the density of the central ring (of the three contained in each image) the density of the central ring is averaged properly, while the density of the surrounding rings is smeared due to their variable spacing. Nevertheless, the spacing of about 80 Å seen in Fig.
7d between the two outer rings suggests that these rings may
be as close together as 40 Å. These side views help establish that the
E1 ring is about 40-50 Å in thickness.
Although the site-specific DNA-binding domains of the
papillomavirus E1 have no homology to the SV-40/polyoma large T
antigens, both of which are highly homologous to each other, and the
nucleotide sequence motifs that serve as binding sites are distinct for
these papillomaviruses; the initiator proteins assemble as helicases in
remarkably similar ways. In both situations a double-ringed structure
assembles at the origin site, and this helicase activity is capable of
denaturing DNA bidirectionally from the assembly point. As we have
shown here the toroidal rings formed by BPV-1 E1 are hexameric as are
the T antigens.
It is also significant to point out that some differences have been
uncovered between these helicases, particularly in the assembly
pathways. Monomers of the T antigens can form hexameric complexes
solely upon incubation with ATP and Mg2+; in contrast, as
we show here E1 must bind DNA in order to assemble as a hexamer.
Similar observations have recently been reported by Sedman and Stenlund
(48), who have shown that single strand DNA can initiate hexamer
formation. That duplex DNA can also affect this multimerization fits
nicely into a pathway through which the enhancer protein E2 helps
target E1 to the ori site and once E2 frees itself from the
preinitiation complex (27) double hexamers may readily follow at
appropriate E1 concentrations.
These apparent biochemical differences may, however, be discussed in
another way that brings the papillomaviruses' mode of DNA replication
even closer to the SV40/polyoma family. For DNA replication in
vivo both SV40 and polyoma large T antigens have enhancer
sequences as cis-dominant elements and for polyoma virus these elements
are absolutely required. Interestingly, the polyomavirus large T
antigen can bind cooperatively with c-Jun, a factor naturally found to
bind to polyoma DNA, and this targeting stimulates helicase activity
(49). Furthermore, E2 can activate polyoma virus replication in the
cell if E2 sites are engineered into viral vectors (50). Although it is
perhaps too premature to speculate on the evolutionary pathway through
which the genes encoding for these viral initiators descend, at least
in part because we do not know how eukaryote chromosomal replication
origins engage or assemble active helicases, it seems possible that the
special relationship that E2 and E1 have with each other mimics
cellular processes captured by the SV40/polyomavirus family.
Image Analysis of the Rings Reveals an Unexpected
Heterogeneity--
Electron microscopy and image analysis have shown
that the rings formed by the E1 protein are hexameric. However, the
population of such rings formed in the presence of an oligonucleotide
are not homogeneous, and two parameters of variability were observed. First, a subset of rings existed not as symmetric hexamers, but as
trimers of dimers, generating a 3-fold rotational symmetry. This has
previously been observed by electron microscopy for the hexameric rings
formed by the dnaB protein (39, 45) and the RecA protein (51). It is
likely that this structural dimerization correlates with the
biochemical non-equivalence of subunits observed for other hexameric
helicases. The T7 gp4 hexamer, for example, has been shown to contain
only three, not six, high affinity ATP-binding sites (52), as has the
hexameric rho protein (53) and DnaB (54).
Second, a large mass of density can exist in the central channel of the
ring, and appears to not depend upon whether the ring has 6- or 3-fold
symmetry. What gives rise to this density? We think it very unlikely
that this density could arise from the oligonucleotide used to induce
ring formation. One primary reason is that this mass appears too large
to be due to the approximately 20-kDa mass of the oligonucleotide. The
mass also appears to be continuously variable in its strength, an
observation not compatible with it arising from the bound
oligonucleotide. Also, since this oligonucleotide is required for ring
formation, it is hard to explain the existence of rings without this
density if the density is due to the oligonucleotide. Third, this mass
appears to be found on the rotational axis of the rings (Fig. 8,
b and d), while we would expect the density due
to the oligonucleotide not only to be much smaller but asymmetrically
displaced from the central axis (10). Based upon our experience imaging
the T7 gp4 helicase with a bound oligonucleotide (10), the weak
asymmetric density that is found within the central channel in the E1
rings shown in Fig. 8, a and c, is consistent
with the density that we would expect from the oligonucleotide.
Since the preparation is at least 95% pure (as judged by SDS-PAGE),
and plugged centers were observed for E1 purified from both E. coli and SF9 cells, and the density is too great to be due to the
oligonucleotide, the most likely explanation is that this central
density arises from a portion of the E1 protein. This density may
therefore arise from a disordered or highly mobile domain of the E1
protein, that can exist in multiple conformations. The recent crystal
structure of an E. coli helicase, the Rep protein (55),
provides a possible clue in this regard. Two copies of Rep were
observed in the crystal, and the two differed by a rotation of the 2B
subdomain by 130°. Since all helicases, including papilloma E1, are
highly likely to have a conserved structure (55, 56), the highly mobile
subdomain in Rep provides support for the possibility that the variable
central density in E1 may be due to the large movement of such a domain.
Observations of the related SV40 large T antigen
helicase3 have also shown a
similar, variable central density. It is noteworthy that a
three-dimensional reconstruction of large T antigen with a hole in the
center (37) only accounted for about 60% of the expected molecular
volume, perhaps due to the fact that a portion of the subunit is mobile
or disordered and not seen in the averaged reconstruction. A recent
study also used electron microscopy to address the multimeric state of
E1. Liu et al. (57) estimated from molecular volumes that E1
complexes on DNA are either hexameric or dihexameric. We did not find
such size heterogeneity on DNA templates and it is possible that the
hexamers observed by Liu et al. were actually intermediates
or aggregates not detected in our experiments.
The Path of DNA through the Double Rings--
The electron
microscopic images of E1 assembled on duplex DNA (Fig. 6) are
consistent with the idea that the DNA somehow passes through the
central cavities of the rings. Our end to end length measurements of
the DNA fragments so engaged with the helicase do not indicate a
shortening and as shown in Fig. 9 this
data by itself would be consistent with two sorts of models for strand passage. In one model both strands might pass through the centers of
the double hexamers as shown in Fig. 9A. In the presence of a single strand binding (SSB) protein and ATP unwinding might proceed
either with the helicase working as a molecular motor translocating
along the DNA and unwinding in opposite directions, or as a molecular
pump denaturing the duplex and forcing it out through a central port.
This class of models is the one that seems to fit most of the data
gathered for the SV40 large T antigen. The DNase I and chemical
protection data (obtained prior to SSB addition) argues in any case
that both strands are protected. Moreover Dean et al. (58)
have shown that preformed hexamers of large T antigen are inactive for
unwinding of circular duplex DNA. We have made similar observations for
the BPV-1 E1 protein (data not shown). These results would be
consistent with models that required a topological link between the
helicase ring and the circular DNA, and that stable hexamers once
formed could not engage the circle.
Curiously, preformed hexamers can displace single-stranded
oligonucleotides annealed to circular single-stranded molecules (58).4 This result perhaps
suggests that a hexameric ring might engage the single strand
circle from an external binding site, track along the DNA and upon
engaging a duplex region pass the other strand through the center of
the ring upon cycles of helicase action. Faced with duplex DNA the
hexamer may have no such entry and therefore a complex assembly process
starting from monomers would create such possibilities for engagement.
Such a model might predict a strand passage situation for duplex DNA as
depicted in Fig. 9B. The electron microscopic data presented
here would also be consistent with this notion. Considerable variation
in lengths for melted or single-stranded DNA have been found, and the
channels created between subunits of the hexamers could space a strand
as close to its complement in this arrangement as in the situation
wherein both strands passed through the center of the rings. (Compare
the positions of the black dots in the cross-sections shown in Fig. 9,
A and B.) In the model shown in Fig.
9B both single strands might be protected from DNase
protection by positing that the external one is buried or wrapped in
the channel. Gillette et al. (59) have concluded from their
results of DNA protection experiments that E1 binding does produce one
type of complex resulting in DNA distortions even in the absence of
ATP. Thus protein binding and assembly of the hexamer around DNA may
provide enough energy to allow for one cycle of denaturation. It is
also possible that the initial complexes depicted in Fig. 9,
A and B, are in some equilibrium with each other
and SSB or ATP might be expected to change this distribution.
Studies with the hexameric replication helicases from E. coli and their T phages have shown that a single subunit contacts the single strand (at a given time) and that the other strand passes
outside of the ring (10, 60). In models for helicase action, this
internal strand might be passed from one subunit to the next in cycles
of ATP hydrolysis. Thus an attraction of the model in Fig.
9B is that it would lead to a conserved mode of action for
the animal viral DNA helicases and their prokaryote relatives.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
opal are
described by Mendoza et al. (16). The BPV-1 origin
containing fragment generated by BamHI and
HindIII restriction enzyme digest of pKSO was inserted into
the pACYC177 vector linearized by BamHI and
HindIII to give rise to pCLO. The 429-base pair BPV-1 origin
containing substrate DNA used in the fragment unwinding assay and for
DNA induced E1 oligomerization was generated by digesting pKSO with the
EcoRI and PvuII restriction endonucleases. The
242-base pair origin containing DNA fragment used in EM linear compaction studies was generated by digesting pKSO with
EcoRI and BamHI restriction endonucleases. Both
of the duplex fragments were purified from agarose gels. The sequence
of the 10-base pair oligomer used for DNA induced E1 oligomerization is
5'-AACAACAATC-3'. The E1 construct used for overexpression in E. coli, pGEX-2TK-E1, was generated by cloning the E1 open reading
frame from the pET11-GST-E1 plasmid (25) into the pGEX-2TK vector
(Pharmacia number 27-4587-01, GenBank accession number U13851). The
integrity of the boundaries for the E1 coding sequence was verified by
DNA sequencing.
-D-galactopyranoside-induced cells
were prepared, cleared of nucleic acid by a Polymin P (10% w/v)
precipitation (0.5% w/v final) centrifuged by a 25,000 × g spin for 20 min. E1 protein in the supernatants was
precipitated by 65% ammonium sulfate at 4° C. The recovered protein
was purified by adsorption to glutathione-Sepharose beads and eluted in
buffer containing 20 nM glutathione. The GST moiety was
cleaved with thrombin and the E1 was further purified by chromatography
on an S-Sepharose column (Pharmacia). The E1 containing fractions were
pooled, dialyzed against E1 dialysis buffer (20 mM
pKPO4, pH 7.5), 150 mM potassium glutamate, 1 mM EDTA, 1 mM DTT, and 10% glycerol aliquoted,
and stored at
80° C.
1), scaled to zero mean density, and the
contrast was normalized. After applying a reference-free alignment
(44), images were ranked by the strength of either the 3- or 6-fold
power, as described in Yu et al. (39). For the 6-fold
ranking, we excluded those images that contained a significant 3-fold
power. After sorting based upon rotational symmetry, images were then
sorted based upon the strength of the integrated density within a 16-Å
radius area at the rotational axis of the ring.
-32P]ATP at 6000 Ci/mM in 20 mM Tris (pH 7.5), 100 mM NaCl, 12 mM MgCl2, and 0.1 mM DTT. The reaction is incubated at 4° C for 30 min and
stopped with the addition of EDTA to 25 mM. E1 was purified away from free 32P and nucleotides by Sephadex G-25 column
chromatography (NAP-10 column, Pharmacia number 17-0854-02).
-mercaptoethanol. The samples were then loaded onto polyacrylamide
gradient gels containing an acrylamide:bis ratio of 80:1. A stacking
phase was not used and electrophoresis was carried out in 25 mM Tris, 250 mM glycine, 0.1% SDS with a final
pH of 8.8. Current was held constant at 10 mA.
RESULTS
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Fig. 1.
Electron micrographs of unwound BPV-1 origin
containing plasmid DNA. Unwound pSS3 DNA molecules cleaved with
either AflII (A and B) or
SacI (C and D) restriction enzymes.
The molecules were applied to carbon grids and rotary shadowed with
tungsten, as described under "Materials and Methods." The
single-stranded DNA appears thicker than duplex DNA, because the
single-stranded DNA is coated with E. coli SSB and/or E1.
Bar in D = 200 nm. E,
measurements of unwinding of BPV-1 origin containing DNA in
vitro. To determine the extent, direction, and location of unwound
regions, molecules were photographed, and the length of duplex DNA was
measured by projecting negatives onto a Numonics digitizing tablet. The
positions of unwound regions (black boxes) were plotted
below a linear map of the plasmid. The relative position of the origin
in base pairs is indicated at the bottom of the histograms.
The standard of deviation for total length was 9%.
OPAL the entire palindrome is deleted. The reaction
products for each were separated by agarose gel electrophoresis, blotted to filters, and probed with pUC18. PhosphorImage analysis of
these Southern blots was used to determine the amount of form U DNA
generated (Fig. 2). The data do show that
while all substrates are capable of directing unwinding the wild type
ori site is indeed preferred. It therefore seems likely that when a
template contains a bona fide ori with E1 sites organized in such a way
as to allow for helicase formation, such sites will be utilized
in vitro. We do not understand why such preferences were not
detected earlier, but perhaps this specificity is sensitive to monomer
E1 concentration and variations in this regard might influence the
data.
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Fig. 2.
E1 helicase prefers origin-containing
substrates. Unwinding reactions were performed with the following
substrates: plasmid pSS3, which has a wild-type E1-binding site,
plasmid pSS3- opal, which lacks the E1-binding site, or plasmid
pSS3-LI5C which has a 5-base pair linker insertion between the inverted
repeats that make up the E1 DNA-binding site. The E1 concentrations
were held constant (368 nM) and
genomic DNA was
titrated into the reactions as nonspecific competitor DNA. The reaction
products were Southern blotted and radioactivity quantitated by
PhosphorImager analysis. The amount of highly unwound form U DNA
generated by helicase activity is plotted. The graph
indicates that substrates with wild-type E1-binding sites are more
effectively unwound than those lacking E1 sites.
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Fig. 3.
Glycerol gradient analysis of E1. 4.5 mg
of purified E1 (1.3 µM final concentration) was incubated
for 10 min at 37° C in 20 mM HEPES (pH 7.5), 50 mM KCl, 7 mM MgCl2, in the presence
or absence of 4 mM ATP. The reactions were subjected to
centrifugation through gradients of 15-35% glycerol as described
under "Materials and Methods." Fractions were collected and
subjected to SDS-PAGE and Western blotting. A Western blot of all
fractions across the gradients is shown. The position of molecular
weight markers run in parallel gradients is indicated above
each protein profile (669 kDa, thyroglobulin; 232 kDa, catalase; 66 kDa, bovine serum albumin). The starting material was loaded in the
lane following the last collected gradient fraction and is labeled
LOAD. The protein profiles of the gradients indicate E1
sediments as two species of approximately 68 and 400 kDa. The material
having a molecular mass greater than 669 kDa is thought to represent
highly aggregated forms of E1. The glycerol gradient fraction
containing the high molecular mass species of E1 (in the presence of
ATP, molecular mass approximately 400 kDa) was applied to
glow-discharged carbon grids and stained with 2% (w/v) uranyl acetate.
The electron micrographs shown were taken at × 30,000 with a JEOL
1200 EX electron microscope at an acceleration voltage of 80 kV.
Bar, 250 Å.
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Fig. 4.
DNA stimulates E1 oligomerization.
A, radiolabled monomeric E1 protein (128 nM) was
incubated in the presence (lanes 6-10) or absence
(lanes 1-5) of a 429-base pair BPV-1 origin containing DNA
fragment (3.8 nM). Glutaraldehyde was titrated into the
reactions (0.005, 0.010, 0.020, and 0.040% final) followed by boiling
in SDS sample buffer + -mercaptoethanol. The reactions were analyzed
by electrophoresis on a denaturing gradient gel (4.4-20% acrylamide)
and stained with Coomassie Brilliant Blue to allow identification of
molecular weight markers. This was followed by drying of the gel and
autoradiography. An autoradiogram of the gel is shown with the
positions of molecular weight markers are indicated on the
left (Kaleidoscope Prestained Standard, Bio-Rad 161-0324)
and right (Cross-Linked Phoshphorylase b, Sigma P
8906). B, glycerol gradient analysis of E1 oligomers induced
by DNA. Radiolabled monomeric E1 (64 nM) was incubated in
the presence or absence of a single-stranded DNA 10-base oligomer
(10-mer, 500 nM) at room temperature for 20 min in 25 mM KPO4 (pH 7.5), 75 mM NaCl, 5 mM EDTA, 1 mM DTT, and 1 mg/ml bovine serum
albumin. Glutaraldehyde was added to a 0.02% final concentration and
incubated for 20 min at room temperature. Glutaraldehyde was removed
from the reactions by Sephadex G-25 column chromatography (NAP-10
column, Pharmacia 17-0854-02). The protein containing fractions were
pooled for each reaction and subjected to centrifugation through
gradients of 15-37% glycerol as described under "Materials and
Methods." Fractions were collected an the amount of radioactivity in
each fraction was determined by scintillation. A graph of the
radioactivity plotted against position in the gradient is shown. The
positions of molecular weight markers run in a parallel gradient is
indicated at the top of the plot. C, the low and
high molecular weight peaks from the glycerol gradients in B
were boiled in SDS sample buffer +
-mercaptoethanol and
analyzed by denaturing gradient gel electrophoresis (4-10%
acrylamide) and stained with Coomassie Brilliant Blue to allow
identification of molecular weight markers. This was followed by drying
of the gel and autoradiography. Two autoradiograms are presented
representing two lengths of exposure time. H (lanes
2 and 4) represents the high molecular weight E1
species isolated from the glycerol gradient in B, and
L (lanes 3 and 5) represents the low
molecular weight E1 peak. The lane marked M (lane
1) is the Coomassie-stained cross-linked phosphorylase
b molecular weight marker (Sigma P 8906) with the weights
indicated to the left. To the right, the
positions of additional molecular weight markers (Kaleidoscope
Prestained Standard, Bio-Rad 161-0324) are indicated.
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Fig. 5.
Oligomerization of E1 correlates with
helicase activity. A, titration of radiolabled E1
monomers (3.75, 7.5, 15, 30, 60, 120, 240, and 480 nM) into
reactions containing 25 mM KPO4 (pH 7.5), 75 mM NaCl, 5 mM EDTA, 1 mM DTT, 1 mg/ml bovine serum albumin, and a 429-base pair BPV-1 origin containing
DNA fragment (4.2 nM) for 20 min at room temperature.
Glutaraldehyde was added to a 0.04% final concentration and incubated
for 20 min at room temperature. Reactions were boiled in SDS sample
buffer + -mercaptoethanol and equal amounts of E1 protein loaded
onto a denaturing acrylamide gradient gel (4-10%). Autoradiography
was performed on the dried gel. The positions of prestained molecular
weight markers are indicated. Cross-linked radiolabled E1 hexamer (408 kDa) purified from a glycerol gradient (see Fig. 6) was loaded as a
mass marker (lane 1). B, fragment unwinding
assay. The helicase assay was performed with the same E1 titration and
DNA levels as above. The DNA fragment is the same as in A
and is radiolabled, while the E1 protein underwent a mock radiolabeling
reaction with cold ATP. The resulting DNA products were assayed by
agarose gel electrophoresis and autoradiography. The lane labeled
boiled provides markers for the ssDNA and double-stranded
DNA positions. C, the amount of denatured DNA was determined
by PhosphorImager analysis and is plotted against E1 protein
concentration.
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Fig. 6.
E1 complex bound to origin DNA.
A, electron micrographs of purified E1 bound to a 242-base
pair DNA fragment containing a centrally located BPV-1 origin sequence.
Samples were prepared and spread as described under "Materials and
Methods." Bar, 100 nM. B,
measurements of DNA lengths with or without bound E1 protein were
performed by projecting electron micrograph negatives onto a Numonics
digitizing tablet. The length distributions of 17 DNA molecules without
E1 and 55 molecules with bound E1 are shown. The distributions are
identical indicating that binding of E1 does not linearly compact the
DNA. C, models for E1 DNA binding. Binding modes are
presented in which the length of DNA would be unchanged upon binding of
a hexameric protein ring. Examples are also provided where the DNA is
wrapped around the helicase ring resulting in linear compaction of the
nucleic acid. The predicted changes in DNA length are shown. These
calculations are based on the dimensions of the E1 hexameric ring
provided by three-dimensional image reconstruction (145 Å maximum
diameter × 50 Å thickness) and the length of the 242-base pair
B-DNA being 823 Å. (standard deviation for DNA length = 9% = 74 Å = 22 base pairs).
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Fig. 7.
Electron micrographs of the E1 protein rings
after incubation with an oligonucleotide (a) or with
double-stranded DNA and ATP (b). The 500-Å scale
bar in a applies to both a and b.
Averaged side views of the rings in b are shown in
c-e. The average in c contains 217 images, while
the subaverages in d and e contain 44 and 52, respectively. The 100-Å scale bar in d applies to
c-e. The double arrow in d is 80 Å long, indicating the approximate spacing between three adjacent
rings.
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Fig. 8.
The E1 rings appear with either a hole in the
center (a and c) or with a plug of
density in the center (b and d).
Independent of whether there is a hole or a plug in the center, the
rings have either a 6-fold symmetry (a and b) or
a 3-fold symmetry (c and d). The images in
e and f are the averages in a and
b, respectively, but with an exact 6-fold symmetry imposed,
while the images in g and h are the averages in
c and d, respectively, but with an exact 3-fold
symmetry imposed. The number of individual ring images in the averages
shown is: a, 400; b, 278; c, 56; and
d, 44. The arrows in a and
c indicate a weak density that is located asymmetrically in
the central channel, consistent with what might be expected from the
bound oligonucleotide. The scale bar in h is 100 Å. The rotational power spectrum (63) for the average of 774 E1 rings
showing a 6-fold rotational symmetry (with no 3-fold) (i),
and the rotational power spectrum for an average of 100 E1 rings
showing a significant 3-fold rotational symmetry (j). The
spectra were calculated between the radial limits of 12 to 68 Å.
DISCUSSION
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Fig. 9.
Models illustrating how hexameric helicases
might engage DNA. A, two hexameric helicases assemble
as rings topologically linked to DNA with both strands passing through
their central cavities. Two possible modes of unwinding are shown. To
the left, the helicases translocate away from each other
along the DNA, leaving single-stranded DNA in their wake. On the
right is a mode of unwinding in which the helicases remain
in contact and the single strands are spooled out between the ring:ring
interface. This arrangement has been proposed for SV40 large T antigen
based on electron microscopy of T antigen-mediated DNA unwinding
reactions and is analogous to the way in which E. coli RuvB
assembles at Holliday junctions (38, 64, 65). For this case a mechanism
for keeping the strands apart within the ring cavity must operate, for
example, two binding sites for single strands (5' to 3' and 3' to 5').
B, an adaptation of the model mentioned above addressing the
possibility that the helicases assemble on DNA with only a single
strand passing through the central cavity of each ring. Note that in
model B a shortening of the DNA may be anticipated, but this
shortening is a function of the angle between the two rings, as well as
the precise contour and width of the rings.
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ACKNOWLEDGEMENTS |
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We thank Arne Stenlund for providing the E. coli expression system for E1, Seth Harris and James Berger for a critical reading of the manuscript, and Terri DeLuca for word processing.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA42414 and CA30490 (to M. B.) and GM35269 (to E. H. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Life Science Div., Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
To whom correspondence should be addressed. Fax: 510-643-6334;
E-mail: mbotchan{at}uclink4.berkeley.edu.
The abbreviations used are: BPV, bovine papiloma virus; GST, glutathione S-transferase; DTT, dithiothreitol; SSB, single stranded-binding protein; PAGE, polyacrylamide gel electrophoresis.
2 C. Sanders, personal communication.
3 X. Yu and E. H. Egelman, unpublished data.
4 E. Fouts and M. R. Botchan, unpublished observations.
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REFERENCES |
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