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J. Biol. Chem., Vol. 275, Issue 29, 22273-22277, July 21, 2000
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,,,¶
From the Department of Medical Genetics and
Microbiology, University of Toronto, Toronto, Ontario M5S 1A8 and the
§ Banting and Best Department of Medical Research,
University of Toronto, Toronto, Ontario M5G 1L6, Canada
Received for publication, February 22, 2000, and in revised form, April 27, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The EBNA1 (for Epstein-Barr nuclear antigen 1)
protein of Epstein-Barr virus governs the replication and partitioning
of the viral genomes during latent infection by binding to specific
recognition sites in the viral origin of DNA replication. The crystal
structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homologous to the DNA
binding domain of the papillomavirus E2 protein, and a flanking domain,
which mediated all the observed sequence-specific contacts. To test the
possibility that the EBNA1 core domain plays a role in
sequence-specific DNA binding not revealed in the crystal structure, we
examined the effects of point mutations in potential hydrogen bond
donors located in an Epstein-Barr virus
(EBV)1 infects most people
worldwide and is associated with several types of cancer (1). The
infection usually takes a latent form in which the viral DNA episomes
are replicated once every cell cycle and are maintained at a constant copy number per cell (1, 2). During latent infection, various combinations of as many as nine viral proteins are expressed, but only
one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1), is expressed
in all cases (3). EBNA1 is the only viral protein required for the
replication and stable maintenance of EBV genomes (4). EBNA1 also
regulates the expression of itself and other latent gene products by
both transactivating and repressing transcription (5-7).
The replication, segregation, and transactivation functions of EBNA1
require the binding of EBNA1 dimers to 18-bp palindromic recognition
sites present in multiple copies in the viral "plasmid" origin of
replication, oriP (8-10). EBNA1 residues that mediate DNA
binding and dimerization have been localized to the C-terminal portion
of the protein between amino acids 459 and 607 (11-14). Mutagenesis of
this region identified residues 459-487 as important for
sequence-specific DNA recognition (13, 14).
The crystal structure of the EBNA1 DNA binding and dimerization region
was solved both in the presence and absence of its 18-bp recognition
site (15-17). These structures revealed that this region comprised two
closely associated domains, referred to as the core domain and the
flanking domain (see Fig. 1). The core domain (amino acids 504-604)
contains an eight-stranded antiparallel Although the EBNA1 co-crystal structure did not reveal a direct role
for the core domain in sequence recognition, the core domain is
hypothesized to play an important role in DNA binding for several
reasons. First, the core domain is structurally homologous (root mean
square deviation 0.908 Å) to the complete DNA binding domain of the E2
protein of bovine papillomavirus; this structural homology suggests a
common function and DNA binding mechanism. In E2, each monomer contains
a helix, termed the recognition helix, which makes all of the base
contacts with the DNA (18). The analogous helix (also termed the
recognition helix) in the EBNA1 core domain contains three amino acids
(Lys514, Tyr518, and Arg522) that
are potential hydrogen bond donors (15) (Fig. 1). Second, the potential
hydrogen bond donors in the EBNA1 recognition helix are preferentially
conserved in the distantly related EBNA1 protein from herpesvirus papio
(19). Third, an EBNA1 DNA binding domain mutant lacking most of the
flanking domain binds some EBNA1 recognition sites with high affinity,
despite the fact that it lacks the residues that mediate five out of
the seven base-specific contacts (12). Finally, weak DNA binding has
been observed with an EBNA1 fragment lacking the complete flanking
domain (11).
In this paper we investigated the role of the EBNA1 core domain in DNA
binding by mutating potentially important hydrogen bond donors in the
core domain and by analyzing the core domain in the absence of the
flanking domain. Our data reveal that the core domain plays a direct
role in DNA recognition.
The EBNA1 Mutants--
EBNA1 mutants with alanine substitutions
in arginine 469 (R469A), tyrosine 518 (Y518A), arginine 522 (R522A), or
lysine 514, tyrosine 518, and arginine 522 (RH) were constructed by
amplifying the EBNA1 gene in two fragments; the 3' end to the mutation
site and the mutation site to the 5' end. The polymerase chain reaction products were phosphorylated and ligated together, and EBNA1 sequences between amino acids 452 and 641 were reamplified using oligonucleotides that placed an NdeI site just N-terminal to residue 452 and
a BamHI site C-terminal to residue 641. The polymerase chain
reaction products were digested with NdeI and
BamHI and cloned between the NdeI and
BamHI sites of pET15b (Novagen). The resulting constructs expressed EBNA1 residues 452-641 (with point mutations) fused to a
hexahistidine tag. All constructs were sequenced to confirm the
positions of the point mutations.
The EBNA1 fragment corresponding to amino acids 496-641 was generated
by polymerase chain reaction amplification using primers that placed an
NdeI site prior to codon 496 and a BamHI site
following codon 641. The EBNA496-641 expression construct
was generated by digesting the amplified EBNA1 fragment with
NdeI and BamHI and ligating it between the
NdeI and BamHI sites of pET15b.
Protein Purification--
EBNA452-641 was
overproduced and purified from Escherichia coli as described
previously (20). R469A, Y518A, R522A, RH, and EBNA496-641
were expressed in E. coli strain BL21(DE3) pLysS and
purified on heparin-agarose and metal chelating (nickel) columns as
described previously for EBNAWF (21). Hexahistidine tags
were removed from the purified proteins by thrombin digestion and
dialysis as described by Summers et al. (21). For
EBNA496-641, following thrombin digestion and dialysis,
the protein was passed through the nickel column to remove any EBNA1
protein that still retained the hexahistidine tag.
EBNA496-641 in the nickel column flow-through was then
concentrated in a Centriprep centrifugal filter unit (Millipore). The
concentrations of the purified proteins were determined from absorbance
readings at 280 nm.
Analysis of Protein Folding and Unfolding--
The secondary
structures of the EBNA1 proteins were compared using an Aviv 62A DS
circular dichroism spectrometer. A 10 µM solution of each
protein in phosphate-buffered saline, pH 7.4, was scanned from 320 to
200 nm at 25 °C in a 0.1-cm cuvette using a 1-s averaging time. For
protein stability studies, concentrated protein samples were rapidly
diluted into guanidine hydrochloride (GdnHCl; Pierce) buffer, and the
loss of circular dichroism (CD) signal over time was monitored at 222 nm, beginning 5-10 s after dilution into GdnHCl. The raw data were
normalized and fit to the equation y = 1 DNA Binding Assays--
DNA binding assays used a single EBNA1
recognition site corresponding to site 1 of the oriP dyad
symmetry element. To generate this site, the 20-mer oligonucleotide
5'-CGGGAAGCATATGCTACCCG-3' was end-labeled with
[ A Role for the EBNA1 Recognition Helix in DNA Binding--
The
EBNA1 core domain contains a helix that is structurally homologous to
the "recognition helix" in E2. In E2, this helix harbors all of the
residues that make base-specific contacts with the DNA. To investigate
the role of the EBNA1 recognition helix, we generated three different
EBNA1 mutants in which potential hydrogen bond donors of the
recognition helix were mutated to alanines (see Fig.
1). In one mutant, called RH, all three
potential hydrogen bond donors were disrupted (Lys514,
Tyr518, and Arg522); in the second mutant,
tyrosine 518 was targeted (Y518A); and the third mutant contained a
point mutation in arginine 522 (R522A). In the EBNA-DNA co-crystal
structure, the three targeted amino acids are oriented toward the DNA
but are too far from the nearest H-bond acceptor in the bases (more
than 6 Å) to form H-bonds (16). Arg522 does, however, form
a H-bond with one phosphate residue.
All three mutations were introduced in the context of the DNA binding
and dimerization region (amino acids 452-641), purified, and titrated
onto an EBNA1 recognition site (Fig. 2
and Table I). The DNA binding activity of
each mutant, as analyzed by electrophoretic mobility shift assays
(EMSAs), was compared with the equivalent wild type EBNA1 polypeptide
(EBNA452-641). All of the mutant proteins showed defects
in DNA binding. The triple mutant and R522A had severely diminished
EBNA1 DNA binding activity, and the activity of the Y518A mutant was
reduced 80-fold compared with the wild type polypeptide. These
experiments were performed three times for each mutant with very
similar results.
To ensure that the mutations in the recognition helix affected DNA
binding specifically rather than disrupted the folding of the protein,
we tested the effects of the mutations on protein folding using CD
spectroscopy. The CD spectra of the three recognition helix mutants
were indistinguishable from the wild type polypeptide (Fig.
3). All four proteins had elliptical
minima at 208 and 222 nm, in keeping with their helical content.
We also tested whether the recognition helix mutations were less active
because they destabilized the EBNA1 dimer. The active form of EBNA1 is
a dimer, and the dimerization interface is highly similar to the E2
protein from papillomavirus (15, 18, 22). In E2, dissociation of the
dimer leads to rapid unfolding and inactivation (23, 24). To test the
effects of the recognition helix mutations on the stability of
the EBNA1 dimer, we monitored the rates of unfolding of the EBNA1
proteins in guanidine using circular dichroism at 222 nm (Table
II). EBNA452-641 was extremely stable, requiring a 33-min incubation in 6.6 M
GdnHCl to unfold half of the protein (standard equilibrium stability assays were not possible because the unfolding of
EBNA452-641 was not reversible). The Y518A mutation had no
significant effect on the stability of the protein. The triple point
mutation had only a small effect on EBNA1 stability, with half of the
protein remaining folded after 14 min in 6.6 M guanidine.
Our data suggest that all of the EBNA1 mutants remained dimeric and
folded during the 10-min DNA binding reaction (in the absence of
guanidine). Our results therefore support a direct role for the EBNA1
recognition helix in DNA recognition.
The Core DNA Binding Domain Is Sufficient for Sequence-specific DNA
Binding--
The above data strongly suggest that the core domain
recognition helices play a direct role in DNA recognition, but
alternative explanations are possible. For example, the recognition
helix mutations could have elicited their effects by causing minor
structural changes that would not be detected by CD analysis or by
disrupting DNA phosphate (as opposed to base) interactions. To directly
examine the contribution of the core domain to DNA binding, we
expressed and purified an EBNA1 polypeptide containing the core domain
but lacking the flanking domain (EBNA496-641), and
monitored sequence-specific DNA binding by EMSAs. Like the EBNA1
proteins examined above, EBNA496-641 contained the
C-terminal acidic tail in addition to DNA binding domain residues (the
EBNA1 core domain produced in the absence of the acidic tail was insoluble).
Purified EBNA496-641 protein was titrated onto two
different DNA fragments, one that contained the EBNA1 binding site and
a second 18-bp sequence that contained the DNA recognition site for the
E2 protein of bovine papillomavirus. As shown in Fig.
4, EBNA496-641 bound to the
EBNA1 site in a saturable manner but not to the E2 site. Thus, the core
domain is capable of sequence-specific DNA binding in the absence of
the flanking domain. The complex formed by EBNA496-641 and
the EBNA1 recognition site was consistently observed to migrate in the
gel as a doublet, possibly reflecting variations in the conformation of
the bound DNA. The affinity of EBNA496-641 for the EBNA1 site was approximately 800-fold less than that observed for
EBNA452-641 (Table I).
The Flanking Domain Contributes to Sequence-specific DNA
Binding--
The crystal structure of EBNA1 bound to DNA showed that
all base contacts were made by flanking domain residues, whereas the current EMSA data indicate that the core domain plays a major role in
base recognition. These differences indicate either: 1) that EBNA1 has
two independent modes of DNA recognition, the flanking domain mode that
occurs under the crystallography conditions and the core domain mode
that occurs in the EMSAs, or 2) that both the core and flanking domains
contribute to DNA binding but that the core domain base contacts are
transient and therefore not observed in the crystal structure. To
distinguish between these two possibilities, it was important to
determine whether flanking domain residues, in the context of the
entire DNA binding region, contributed to the DNA binding activity
detected by EMSAs. The reduced DNA binding affinity of
EBNA496-641 relative to EBNA452-641 suggested
that the flanking domain was contributing to DNA binding but could
conceivably be due to a reduction in the percentage of the protein that
is folded.
To determine the contribution of the flanking domain to DNA binding
activity of EBNA452-641, we mutated arginine 469 to
alanine within the context of amino acids 452-641 (R469A). In the
EBNA1 co-crystal structure, arginine 469 makes two base contacts and is
located in the extended chain that sits in the DNA minor groove (16).
R469A was expressed, purified, and assayed for binding to the EBNA1
recognition site exactly as performed with the recognition helix
mutants. As shown in Fig. 5 and Table I,
the mutation of R469 reduced DNA binding activity approximately 300-fold. As was observed for EBNA496-641, the DNA complex formed by R469A migrated as a doublet.
The crystal structure of the EBNA1 DNA binding region bound to DNA
revealed two domains: the core domain, which mediated dimerization, and
the flanking domain, which mediated base contacts with the DNA (16). We
have now shown that the core domain also plays a direct role in
sequence-specific DNA interactions via two A second explanation is that both the core and flanking domains mediate
DNA contacts under the EMSA and crystallography conditions, but that
the recognition helices are dynamically associated with the DNA. In
this case, the crystal structure might simply have captured the
recognition helices in the dissociated position. Based on the
co-crystal structures of EBNA1 and E2, the base contacts made by the
EBNA1 recognition helices are expected to occur through the major
groove. The possibility that the core and flanking domains contact the
DNA at the same time is supported by methylation protection footprints
that show protection of a G residue (at position 3/ The third possibility is that EBNA1 binds DNA by a two-step mechanism
in which both the core and flanking domains interact sequentially with
the EBNA1 recognition site. In this mechanism, the core domain
recognition helices would make the first sequence-specific DNA
contacts, positioning the protein such that the flanking domain extended chain can be loaded into the minor groove. After the flanking
domain is assembled on the DNA, the position of the recognition helices
would shift such that direct contacts with the bases are no longer
stable. Thus, in the assembled EBNA1-DNA complex that was crystallized,
no base contacts with the core domain would be observed. During
dissociation of EBNA1 from the DNA, the recognition helices might again
make transient DNA contacts, facilitating the release of the flanking
domain from the DNA.
A key feature of models two and three is that the recognition helices
and the flanking domain extended chain are flexible. Evidence for the
former comes from NMR studies on the E2 DNA binding domain, which
revealed that the E2 recognition helices are mobile (26). The
flexibility of the EBNA1 flanking domain extended chain was revealed in
the crystal structure of EBNA470-607 in the absence of DNA
(15). In this structure, the extended chains in the two monomers were
disordered. Thus, DNA binding models two or three are likely, and
further kinetic analyses will be necessary to differentiate between the two.
Since EBNA1 dimerization is a requirement for DNA binding, we have
addressed the possibility that the recognition helix mutations affect
DNA binding indirectly by destabilizing the dimerization of the
protein. In these experiments, we used protein unfolding as a measure
of disruption of the dimerization interface. Several lines of evidence
indicate that unfolding will occur if the dimerization of EBNA1 is
disrupted. First, the EBNA1 core domain that mediates dimerization is
almost identical in structure to the DNA binding and dimerization
domain of the papillomavirus E2 protein. Careful studies on the folding
of E2 have clearly shown that dissociation of the dimer only occurs
when the domain unfolds (23, 24). Second, the recognition helix point
mutations are distant from the residues in the four DNA binding mechanisms involving two DNA interaction modes or steps are
unusual but not completely novel. For example, the Myb oncoprotein and
T7 and E. coli RNA polymerases have some dynamic characteristics. Tryptophan fluorescence studies indicate that the
initial rapid interaction of Myb with DNA is followed by a slower step,
which involves a structural reorganization of the protein (27).
Similarly, kinetic studies with T7 RNA polymerase indicate that this
protein forms an initial weak complex with promoter DNA, then undergoes
a conformational changes resulting in tighter DNA binding (28). The
multisubunit E. coli RNA polymerase has at least two
biochemically defined nucleic acid binding domains that interact
transiently during the course of the polymerization reaction and
stabilize the ternary elongation complex (29-31); the dynamic nature
of these interactions underlies the regulation of RNA synthesis.
Perhaps the dynamics of the interaction of the EBNA1 domains with DNA
also plays a regulatory role in the initiation of DNA replication.
-helix of the EBNA1 core domain whose
structural homologue in E2 mediates sequence-specific DNA binding. We
show that these mutations severely reduce the affinity of EBNA1 for its
recognition site, and that the core domain, when expressed in the
absence of the flanking domain, has sequence-specific DNA binding
activity. Flanking domain residues were also found to contribute to the
DNA binding activity of EBNA1. Thus, both the core and flanking domains
of EBNA1 play direct roles in DNA recognition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel, comprised of four
strands from each monomer and two -helices per monomer. The core
domain forms the dimerization interface but was not observed to mediate
contacts with the DNA bases. The flanking domain (amino acids 461-503)
comprises an -helix and an extended chain, and mediates all of the
base contacts observed in the co-crystal structure. The helix is
oriented perpendicular to the axis of the DNA and its N-terminal amino
acid (Lys477) contacts two bases in the outer portion of
the DNA recognition site through the major groove. Five additional base
contacts are made by three amino acids (Lys461,
Gly463, and Arg469) in a portion of the
extended chain that tunnels along the base of the minor groove.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
exp(kt) using the Kaleidograph program.
-32P]ATP and then annealed to its complementary
oligonucleotide. For equilibrium binding assays, EBNA1 proteins were
incubated with 10 fmol of end-labeled DNA recognition site for 10 min
at room temperature in 20-µl reactions containing 10 mM
HEPES (pH 7.5), 5 mM MgCl2, 300 mM
NaCl (binding buffer). Glycerol was then added to a final concentration
of 5%, and the reactions were loaded onto a 12% polyacrylamide gel
containing 0.5× TBE. After electrophoresis, bound and unbound DNA was
visualized by autoradiography and quantified using a
PhosphorImager (Molecular Dynamics) and Imagequant software. The
ability of EBNA496-641 to bind to the recognition
site of the papillomavirus E2 protein was also tested as described above for the EBNA1 recognition site. The E2 recognition site was
generated by annealing the end-labeled, palindromic 18-mer 5'-CCGACCGACGTCGGTCGG-3' to itself.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (71K):
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Fig. 1.
Structure of the EBNA1 DNA binding and
dimerization region. The crystal structure of EBNA1 residues
461-607 bound to an 18-bp EBNA1 recognition site was solved previously
by Bochkarev et al. (16) (Protein Data Bank entry 1B3T). The
flanking domain is shown in red and the core domain in
blue; the recognition helices are shown in a
darker shade of blue to distinguish
them from the rest of the core domain. Side chains of amino acids 514 (Lys), 518 (Tyr), and 522 (Arg) in the recognition helix are also
shown.
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Fig. 2.
Equilibrium DNA binding assays.
Wild type (EBNA452-641) and mutant EBNA1 proteins were
titrated with an end-labeled EBNA1 recognition site, and bound and free
DNA fragments were separated by acrylamide gel electrophoresis.
Dissociation constants of EBNA1 proteins on the EBNA1 recognition
site
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Fig. 3.
Analysis of protein structure by circular
dichroism. CD spectra of Y518A, R522A, and RH mutants were
compared with the EBNA452-641 wild type protein at 10 µM protein concentration.
Summary of protein unfolding experiments in 6.6 M guanidine
View larger version (72K):
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Fig. 4.
Sequence-specific DNA binding activity of
EBNA496-641. EBNA496-641 was titrated
onto end-labeled DNA fragments containing either an EBNA1 or an E2
recognition site. Bound and free DNA fragments were separated by
acrylamide gel electrophoresis.
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Fig. 5.
The arginine 469 flanking domain residue is
important for DNA binding. The R469A EBNA1 mutant was titrated
onto an EBNA1 recognition site, and binding was compared with wild type
EBNA1 (EBNA452-641) by electrophoretic mobility shift
assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices termed the
recognition helices. We envisage three possible ways in which the
biochemical and structural data on the EBNA1-DNA interaction can be
reconciled. First, EBNA1 may have two different modes of DNA binding:
one that involves the flanking domain and a second that uses the core
domain. In this scenario, the flanking domain binding mode would be
favored under the conditions of the crystallography, whereas the core
domain binding mode would be favored under the conditions of the
binding reactions for the EMSAs. This explanation is unlikely because
point mutations both in the core domain recognition helices and in the
flanking domain extended chain have pronounced effects on DNA binding
under the conditions of the EMSAs. Furthermore, the crystal structure
of the EBNA1 DNA binding region has been solved on three different EBNA1 recognition sequences (including the site 1 sequence studied here) under varied salt and buffer conditions and, in all cases, the
hydrogen bonds observed between EBNA1 residues and the DNA bases were
identical (16, 17).2
3 relative to the
axis of symmetry of the palindrome) that is not contacted by flanking
domain residues in the crystal structure but is predicted to be
contacted by Lys514 in the recognition helix (16, 25).
-sheets known to
mediate dimerization, and therefore any effects on dimerization would
be expected to involve destabilization of the domain (15). Third, EBNA1
is only found as a dimer under native conditions; folded monomers of
EBNA1 have never been observed (10). Finally, the dimeric interface
forms the hydrophobic core of the domain and, thus, folding of an EBNA1
monomer is energetically very unlikely. We believe, therefore, that the
defect associated with the EBNA1 recognition helix mutations is in DNA
binding and not in dimerization. One caveat to our EBNA1 dimerization
analysis was that the CD experiments were only conducted at one protein
concentration (10 µM); therefore, we have not ruled out
the possibility that the EBNA1 dimers dissociate at lower protein
concentrations. However, the DNA binding analyses of the RH and R522A
mutants were conducted at protein concentrations similar to those used
in the CD analysis (up to 26 µM for RH and up to 6 µM for R522A); therefore, the DNA binding defects
exhibited by these mutants are unlikely to be due to disruption of dimerization.
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ACKNOWLEDGEMENTS |
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We thank Angela Fleming for the construction and purification of the R469A EBNA1 mutant, and Valerie Booth for generating Fig. 1. We also thank Karen Maxwell and members of the Davidson laboratory for helpful discussions on protein folding.
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FOOTNOTES |
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* This work was supported by a grant (to L. F. and A. M. E.) from the National Cancer Institute of Canada, which receives funds from the Canadian Cancer Society.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.
¶ Medical Research Council of Canada Scientist.
To whom correspondence should be addressed: Dept. of Medical
Genetics and Microbiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-946-3501; Fax:
416-978-6885; Email: lori.frappier@utoronto.ca.
Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M001414200
2 A. Bochkarev, A. M. Edwards, and L. Frappier, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: EBV, Epstein-Barr virus; bp, base pair(s); EMSA, electrophoretic mobility shift assay; GdnHCl, guanidine hydrochloride.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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