Biomedical subjects

L Frappier

Publications and source records attributed to L Frappier.

34 records · Page 2Linked to original sources

PubMed1996

Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein, EBNA1, bound to DNA.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP, the latent origin of DNA replication in Epstein-Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1-origin interaction.

Amino Acid Sequence↗

PubMed1996

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.

Antigens, Viral↗

PubMed1995

Identification of Epstein-Barr virus nuclear antigen 1 protein domains that direct interactions at a distance between DNA-bound proteins.

The EBNA1 protein of Epstein-Barr virus (EBV) binds to and activates DNA replication from the EBV latent origin of replication, oriP, via a direct interaction with the two noncontiguous subelements of oriP. The EBNA1 molecules bound to the oriP subelements interact efficiently with each other by a DNA looping mechanism. We have previously mapped a region of EBNA1 (termed the looping region) that is required to mediate the interaction of the EBNA1 molecules bound to the oriP subelements. We now demonstrate that two fragments of this region of EBNA1, which consist largely of an eight amino acid repeat, can mediate homotypic interactions when transferred to another DNA-binding protein. Protein interactions mediated by the EBNA1 looping region appear to be dependent on DNA binding since these interactions were detected between DNA-bound forms of the proteins only.

Amino Acid Sequence↗

PubMed1995

Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein EBNA 1.

The crystal structure of the DNA-binding and dimerization domains of the Epstein-Barr virus nuclear antigen 1 (EBNA1), which binds to and activates DNA replication from the latent origin of replication in Epstein-Barr virus, was solved at 2.5 A resolution. EBNA1 appears to bind DNA via two independent regions termed the core and the flanking DNA-binding domains. The core DNA-binding domain, which comprises both the dimerization domain and a helix predicted to bind the inner portion of the EBNA1 DNA recognition element, was remarkably similar to the structure of the papillomavirus E2 protein, despite a complete lack of sequence conservation. The flanking DNA-binding domain, only a portion of which is contained in the current structure, consists in part of an alpha helix whose N-terminus contacts the outer regions of the EBNA1 DNA recognition element.

Amino Acid Sequence↗

PubMed1995

Overexpression, purification, and crystallization of the DNA binding and dimerization domains of the Epstein-Barr virus nuclear antigen 1.

The Epstein-Barr virus nuclear antigen (EBNA) 1 binds to and activates DNA replication from the latent origin of Epstein-Barr virus. Six different fragments of EBNA1 that retain DNA binding activity were expressed in bacteria, purified, and crystallized. Two fragments, EBNA470-619 and EBNA470-607, formed well ordered crystals that diffracted beyond 2.5-A resolution. Two different EBNA470-619 crystals were grown from sodium formate, pH 6-6.5. One crystal belonged to the trigonal space group P3 with unit cell dimensions a = b = 86.5 A and c = 31.8 A and with two molecules in the asymmetric unit. The other crystal, which appeared only twice and was likely related to the P3 crystal form, belonged to the trigonal space group P312 with cell dimensions a = b = 86.7 A and c = 31.8 A. Crystals of EBNA470-607 were grown by lowering the salt concentration to 0-100 mM NaCl at pH 6.0. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and had cell dimensions a = 59 A, b = 66.9 A, and c = 69.8 A with two molecules in the asymmetric unit.

Antigens, Viral↗

PubMed1994

Stabilization of the EBNA1 protein on the Epstein-Barr virus latent origin of DNA replication by a DNA looping mechanism.

DNA replication from the Epstein-Barr virus latent origin of replication, oriP, is activated by Epstein-Barr nuclear antigen 1 (EBNA1). This activation involves the binding of EBNA1 dimers to multiple sites present in the two noncontiguous functional elements of oriP, the dyad symmetry element (DS) from which replication initiates, and the family of repeats (FR) enhancer element. EBNA1 complexes formed on the FR and DS elements of oriP interact by a DNA looping mechanism. This interaction requires EBNA1 sequences in addition to those required for DNA binding and dimerization. To map the EBNA1 sequences required for the efficient interaction of FR- and DS-bound EBNA1 complexes, we have overproduced in Escherichia coli and purified a series of EBNA1 N-terminal truncation mutants, all of which retain the DNA binding and dimerization domains. The results of electron microscopy and ligation-enhancement assays using these mutants indicated that EBNA1 sequences between amino acids 350 and 361 are required for the efficient interaction of FR- and DS-bound EBNA1 complexes. EBNA1-mediated FR-DS interactions were shown to stabilize EBNA1 binding to the DS element, while EBNA1-mediated DS-DS interactions did not. These results suggest that the stabilization of EBNA1 on the DS element, which occurs as a result of EBNA1-mediate oriP looping, may be important for the activation of DNA replication from the DS element.

Antigens, Viral↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1992

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.

Antigens, Viral↗

PubMed1991

Epstein-Barr nuclear antigen 1 mediates a DNA loop within the latent replication origin of Epstein-Barr virus.

Epstein-Barr virus-encoded nuclear antigen 1 (EBNA-1) binds and activates the viral latent origin of DNA replication, oriP. We have used electron microscopy to examine the assembly of EBNA-1 onto oriP. The oriP region consists of two essential elements separated by approximately 1 kilobase pair of DNA. One element contains 20 tandom EBNA-1 binding sites [called the family of repeats (FR)] and serves to activate initiation of replication at the dyad symmetry (DS) element, which contains 4 EBNA-1 binding sites. Titration of homogeneous EBNA-1 produced in baculovirus (bEBNA-1) onto oriP DNA showed an order to the assembly of bEBNA-1 onto oriP. At low concentrations, bEBNA-1 was located exclusively on the FR element. As the level of bEBNA-1 was raised, a loop between the FR and DS elements became the most prevalent DNA-protein complex. These data suggest protein-mediated DNA looping may play a role in activating latent-phase replication of the Epstein-Barr virus.

Antigens, Viral↗

PubMed1991

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.

Animals↗

PubMed1991

Overproduction, purification, and characterization of EBNA1, the origin binding protein of Epstein-Barr virus.

The baculovirus expression system was used to overproduce the Epstein-Barr virus nuclear antigen, EBNA1, in insect cells. EBNA1 overproduced via baculovirus expression (baculoEBNA1) was followed during purification to homogeneity using its ability to specifically retain the family of repeats of the latent origin of replication, oriP, onto nitrocellulose filters. A two-column procedure was developed which yields more than 1 mg of homogeneous baculoEBNA1 from 9 x 10(8) insect cells (1.5 liters). Pure baculoEBNA1 had no detectable ATPase or helicase activity. BaculoEBNA1 was labeled with [32P]orthophosphate in vivo, and analysis showed detectable levels of phosphoserine; no phosphothreonine or phosphotyrosine could be detected. The baculoEBNA1 appeared dimeric in solution, and a stoichiometry of 56 baculoEBNA1 monomers per 24 EBNA1 binding sites in oriP suggests baculoEBNA1 binds its consensus site as a dimer. The binding of baculoEBNA1 to the dyad symmetry element of oriP (Kd approximately 2 nM) required more baculoEBNA1 and appeared less stable than the binding of baculoEBNA1 to the family of repeats in oriP (Kd approximately 0.2 nM).

Amino Acids↗

PubMed1989

Characterization of the binding specificity of two anticruciform DNA monoclonal antibodies.

Two monoclonal antibodies (2D3 and 4B4) have been raised against a stable cruciform DNA structure containing the 27-base pair palindrome of the SV40 origin of replication on one strand and an unrelated 26-base pair palindrome on the complementary strand (pRGM 21 x pRGM 29) and have been shown to recognize conformational determinants specific to cruciform DNA structures (Frappier, L., Price, G.B., Martin, R. G., and Zannis-Hadjopoulos, M. (1987) J. Mol. Biol. 193, 751-758). To define the region(s) of the cruciform that is recognized by these antibodies, we examined the ability of 2D3 and 4B4 to protect the single-stranded tips of the loops or the four-way junctions at the base of the stem of stable cruciform molecules against cleavage by mung bean nuclease or T7 endonuclease 3, respectively. Both antibodies were found to protect two of the four elbow-like structures at the base of the cruciform from T7 endonuclease 3 cleavage, but not the tips of the cruciform arms from mung bean nuclease cleavage. Also, predigestion of the cruciform with mung bean nuclease did not affect the binding of either antibody. In addition, 2D3 bound to a cruciform and a T-shaped structure involving the palindromic sequence at the cloning site of pUC7, which is completely unrelated in sequence to the palindrome of pRGM 21 x pRGM 29, and protected the base of these stem-loop structures against cleavage by T4 endonuclease VII. These results indicate that 2D3 and 4B4 bind at or near the base of the cruciform molecules and that, at least for 2D3, binding is independent of DNA sequence.

Antibodies, Monoclonal↗

PubMed1988

Effect of anti-cruciform DNA monoclonal antibodies on DNA replication.

To study the possible involvement of DNA cruciforms in the initiation of DNA replication, we used two monoclonal antibodies, 2D3 and 4B4, with anti-cruciform DNA specificity. Synchronized CV-1 cells were released into S phase for hourly intervals up to 6 h and permeabilized in the presence of monoclonal antibodies, under conditions that allow limited DNA replication. Exposure of the permeabilized cells to 2D3 or 4B4 resulted in a 2- to 6-fold enhancement of incorporation of labeled precursor nucleotide over the 6 h period. Approximately 50% of the enhanced synthesis was sensitive to aphidicolin, and the enhancing effect of 2D3 was abolished by absorption with immunobead anti-mouse immunoglobulin. Dot-blot hybridization analyses of DNA isolated from anti-cruciform antibody treatment groups showed a similar 2- to 11-fold increase in the relative copy number of low copy probes. In contrast, exposure of the permeabilized cells to a monoclonal antibody directed against Z-DNA and B-DNA had no significant effect on DNA synthesis. The results suggest that cruciforms are present in replicating DNA and that they are recognized and stabilized by the monoclonal antibodies.

Animals↗

PubMed1987

Monoclonal antibodies to cruciform DNA structures.

Two monoclonal antibodies, 2D3 and 4B4, have been raised against a cruciform structure in a heteroduplex DNA molecule. Antibody binding to DNA fragments was determined by a radioimmunoassay in which DNA--antibody complexes were separated from unbound DNA by acrylamide gel electrophoresis. These antibodies seem to recognize conformational determinants specific to cruciform structures. 2D3 and 4B4 antibodies do not bind to linear double-stranded homoduplex DNA fragments, linear single-stranded DNA or single-stranded simian virus 40 DNA containing a stem--loop structure, but do bind to the original cruciform and to a different cruciform with one shortened arm. 2D3 also bound to a T-shaped double-stranded DNA molecule, while 4B4 binding to this structure was weak. The monoclonal antibodies 2D3 and 4B4 were found to be immunoglobulin G1 and immunoglobulin M, respectively.

Animals↗

PubMed1987

Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences.

Twelve clones of origin-enriched sequences (ORS) isolated from early replicating monkey (CV-1) DNA were examined for transient episomal replication in transfected CV-1, COS-7, and HeLa cells. Plasmid DNA was isolated at time intervals after transfection and screened by the Dpn I resistance assay or by the bromodeoxyuridine substitution assay to differentiate between input and replicated DNA. We have identified four monkey ORS (ORS3, -8, -9, and -12) that can support plasmid replication in mammalian cells. This replication is carried out in a controlled and semiconservative manner characteristic of mammalian replicons. ORS replication was most efficient in HeLa cells. Electron microscopy showed ORS8 and ORS12 plasmids of the correct size with replication bubbles. Using a unique restriction site in ORS12, we have mapped the replication bubble within the monkey DNA sequence.

Animals↗

PubMed1984

Changes in the cytoplasmic and nuclear activities of the ribonucleotide reductase holoenzyme and its subunits in regenerating liver cells in normal and thyroparathyroidectomized rats.

The level of the cytoplasmic ribonucleotide reductase nonheme-iron-containing L2 subunit in regenerating rat liver cells began rising about 2 h before the onset of DNA synthesis, rose sharply to a maximum level about 4 h before the DNA-synthetic activity reached its peak, and then stayed at this high level even after the cells had finished replicating their DNA. The cytoplasmic level of the CDP-specific, effector-binding L1 subunit and the holoenzyme activity began rising together about 2 h after the L2 subunit began increasing and at the same time as the DNA-synthetic activity, but subsequently rose much more slowly than the L2 subunit and continued rising even after the cells had finished making DNA. The nuclear level of the L2 subunit did not rise in the regenerating liver cells, but the nuclear level of the L1 subunit and the holoenzyme activity began rising together about the same time as the DNA-synthetic activity, peaked briefly 4-6 h before the peak DNA-synthetic activity, and dropped sharply back to the basal levels by the time the DNA-synthetic activity reached its peak, but then rose again slowly as the cells finished making DNA. Thyroparathyroidectomy 72 h before partial hepatectomy prevented the cytoplasmic and nuclear subunits and holoenzyme activity from rising and prevented most of the remaining liver cells from initiating DNA synthesis.

Animals↗