Two domains of the epstein-barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding

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 alpha-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.     

Cooperative assembly of EBNA1 on the Epstein-Barr virus latent origin of replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates DNA replication by binding to multiple copies of its 18-bp recognition sequence present in the Epstein-Barr virus latent origin of DNA replication, oriP. Using electrophoretic mobility shift assays, we have localized the minimal DNA binding domain of EBNA1 to between amino acids 470 and 607. We have also demonstrated that EBNA1 assembles cooperatively on the dyad symmetry subelement of oriP and that this cooperative interaction is mediated by residues within the minimal DNA binding and dimerization domain of EBNA1.     

Replication from oriP of Epstein-Barr virus requires exact spacing of two bound dimers of EBNA1 which bend DNA

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports replication and stable maintenance of plasmids in human cells that contain EBV-encoded protein EBNA1. Plasmids that depend on oriP are replicated once per cell cycle by cellular factors. The replicator of oriP is an approximately 120-bp region called DS which depends on either of two pairs of closely spaced EBNA1 binding sites. Here we report that changing the distance between the EBNA1 sites of a functional pair by inserting or deleting 1 or 2 bp abolished replication activity. The results indicated that, while the distance separating the binding sites is critical, the specific nucleotide sequence between them is unlikely to be important. The use of electrophoretic mobility shift assays to investigate binding by EBNA1 to the sites with normal or altered spacing revealed that EBNA1 induces DNA to bend significantly when it binds, with the center of bending coinciding with the center of binding. EBNA1 binding to a functional pair of sites which are spaced 21 bp apart center to center and which thus are in helical phase induces a larger symmetrical bend, which based on electrophoretic mobility approximates the sum of two separate EBNA1-induced DNA bends. The results imply that replication from oriP requires a precise structure in which DNA forms a large bend around two EBNA1 dimers.     

Kinetically driven refolding of the hyperstable EBNA1 origin DNA-binding dimeric beta-barrel domain into amyloid-like spherical oligomers

The Epstein-Barr nuclear antigen 1 (EBNA1) is essential for DNA replication and episome segregation of the viral genome, and participates in other gene regulatory processes of the Epstein-Barr virus in benign and malignant diseases related to this virus. Despite the participation of other regions of the protein in evading immune response, its DNA binding, dimeric beta-barrel domain (residues 452-641) is necessary and sufficient for the main functions. This domain has an unusual topology only shared by another viral origin binding protein (OBP), the E2 DNA binding domain of papillomaviruses. Both the amino acid and DNA target sequences are completely different for these two proteins, indicating a link between fold conservation and function. In this work we investigated the folding and stability of the DNA binding domain of EBNA1 OBP and found it is extremely resistant to chemical, temperature, and pH denaturation. The thiocyanate salt of guanidine is required for obtaining a complete transition to a monomeric unfolded state. The unfolding reaction is extremely slow and shows a marked uncoupling between tertiary and secondary structure, indicating the presence of intermediate species. The Gdm.SCN unfolded protein refolds to fully soluble and spherical oligomeric species of 1.2 MDa molecular weight, with identical fluorescence centre of spectral mass but different intensity and different secondary structure. The refolded spherical oligomers are substantially less stable than the native recombinant dimer. In keeping with the substantial structural rearrangement in the oligomers, the spherical oligomers do not bind DNA, indicating that the DNA binding site is either disrupted or participates in the oligomerization interface. The puzzling extreme stability of a dimeric DNA binding domain from a protein from a human infecting virus in addition to a remarkable kinetically driven folding where all molecules do not return to the most stable original species suggests a co-translational and directional folding of EBNA1 in vivo, possibly assisted by folding accessory proteins. Finally, the oligomers bind Congo red and thioflavin-T, both characteristic of repetitive beta-sheet elements of structure found in amyloids and their soluble precursors. The stable nature of the "kinetically trapped" oligomers suggest their value as models for understanding amyloid intermediates, their toxic nature, and the progress to amyloid fibers in misfolding diseases. The possible role of the EBNA1 spherical oligomers in the virus biology is discussed.     

Mechanistic studies on the DNA linking activity of Epstein-Barr nuclear antigen 1.

The DNA replication, plasmid segregation and transactivation functions of Epstein-Barr nuclear antigen 1 (EBNA1) require the binding of EBNA1 to specific DNA recognition sites in the two non-contiguous functional elements of the Epstein-Barr virus latent origin of replication, oriP . EBNA1 molecules bound to these elements interact with each other resulting in the formation of looped individual DNA molecules and multiply linked DNA molecules. We have developed a glycerol gradient sedimentation assay suitable for quantitative analysis of the DNA linking activity of EBNA1 and used it to investigate the contribution of EBNA1 residues to the linking interaction and the mechanism of the interaction. Using overlapping internal deletion mutants, we found that two regions of EBNA1 can cause DNA linking, amino acids 40-100 and 327-377, but that the stabilities of the linked complexes formed by the two regions differ dramatically; only complexes formed through the latter region are stable to glycerol gradient sedimentation analysis. Mechanistic studies using EBNA1 in combination with GAL4-EBNA1 fusion proteins showed that linking interactions mediated by residues 327-377 are homotypic. Our results also suggest that only the DNA-bound form of EBNA1 participates in the protein-protein interactions seen in DNA linking.     

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.     

EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient replication of oriP plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.     

Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure.

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607. To identify and define the subdomains for these two functions, we created an extensive series of deletions and point mutations in an EBNA-1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to heterodimerize with a wild-type EBNA-1 (aa 459 to 641) Immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.     

EBNA1 distorts oriP, the Epstein-Barr virus latent replication origin.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed.     

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.     

Inhibition of Epstein-Barr virus OriP function by tankyrase, a telomere-associated poly-ADP ribose polymerase that binds and modifies EBNA1

Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knock-down of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.     

Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose

The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.     

Identification of EBNA1 amino acid sequences required for the interaction of the functional elements of the Epstein-Barr virus latent origin of DNA replication.

Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin, oriP. This activation involves the direct interaction of EBNA1 dimers with multiple sites within the two noncontiguous functional elements of the origin, the family of repeats (FR) element and the dyad symmetry (DS) element. The efficient interaction of EBNA1 dimers bound to these two elements in oriP results in the formation of DNA loops in which the FR and DS elements are bound together through EBNA1. In order to elucidate the mechanism by which EBNA1 induces oriP DNA looping, we have investigated the DNA sequences and EBNA1 amino acids required for EBNA1-mediated DNA looping. Using a series of truncation mutants of EBNA1 produced in baculovirus and purified to apparent homogeneity, we have demonstrated that the EBNA1 DNA binding and dimerization domain is not sufficient to mediate oriP DNA looping and that an additional region(s) located between amino acids 346 and 450 is required. Single EBNA1-binding sites, separated by 930 bp of plasmid DNA, were also shown to support EBNA1-mediated looping, indicating that the formation of large EBNA1 complexes, such as those observed on oriP FR and DS elements, is not a requirement for looping.