DNA looping between the origin of replication of Epstein-Barr virus and its enhancer site: stabilization of an origin complex with Epstein-Barr nuclear antigen 1.

Abstract

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support replication of Epstein-Barr virus during the latent phase of its life cycle. The DNA segment required for latent replication, oriP, contains two essential binding regions for EBNA-1, termed FR and DS, that are separated by 1 kilobase pair. The FR site appears to function as a replicational enhancer providing for the start of replication at the DS site. We have used electron microscopy to visualize the interaction of EBNA-1 with its binding sites and to study the mechanism for communication between the FR and DS sites. We have found that DNA-bound EBNA-1 forms a DNA loop between the FR and DS sites. From these results, we suggest that EBNA-1 bound to the replicational enhancer acts by a DNA-looping mechanism to facilitate the initiation of DNA replication. Occupancy of the DS site alone is highly sensitive to competition with nonspecific DNA. In contrast, occupancy of the DS site by looping from FR is largely resistant to the competitor DNA. These experiments support the concept that enhancers act in cis from nearby sites to provide a high local concentration of regulatory proteins at their target sites and to stabilize regulatory interactions. transcriptional regulation, recent work has demonstrated association ofDNA-bound proteins between sites involved in enhancer function by the bacterial NtrC protein (15), the mammalian Spl protein (16,17), the viral bovine papilloma E2 protein (18), and the Spl and E2 proteins (19). Our work on EBNA-1 has been directed toward two questions: Does replicational enhancement involve the protein-protein association ofDNA-bound EBNA-1? Why do enhancers typically act only from relatively close sites on the same DNA molecule?In the work reported here, we have used electron microscopy to study the interaction of EBNA-1 with its specific binding sites and to examine the ability of DNA-bound EBNA-1 to carry out protein-protein interactions. Our results demonstrate that EBNA-1 bound at the FR and DS sites associates to loop the intervening DNA. As reported in the accompanying paper, Frappier and O'Donnell (20) have also used electron microscopy to show this looping interaction. Based on the sensitivity of the EBNA-1 interaction at the DS site to competition with nonspecific DNA, we suggest that EBNA-1 acts in cis from the FR enhancer to stabilize the interaction of the protein at the DS site. Epstein-Barr virus (EBV) can establish a latent state in which the EBV genome is maintained as a circular plasmid (1, 2). Studies with plasmids derived from EBV DNA have established the functional requirements for replication during the latent state. The Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required for replication from the EBV latent origin of replication, oriP (3, 4). The oriP region is composed of two essential segments separated by about 1 kilobase pair (kbp): (i) a family of repeats (FR) with 20 tandem copies of a 30-bp sequence and (ii) a dyad symmetry region (DS) with 4...

Epstein-Barr Virus Nuclear Antigen 1 Forms a Complex with the Nuclear Transporter Karyopherin α2

“…At this point, it remains unclear whether the transport of both RAG1 and EBNA1 by the nuclear transporter Rch1 and the stimulation of RAG1 (and RAG2) expression by EBNA1 is coincidental. EBNA1 has an intrinsic property to bind to DNA, and it is known that EBNA1 can juxtapose segments of DNA that are otherwise apart from each other (49,54). It is conceivable that EBNA1 might be involved in the formation of DNA complexes that undergo illegitimate recombination as observed in Burkitt's lymphoma.…”

An enhanced EBNA1 variant with reduced IR3 domain for long-term episomal maintenance and transgene expression of oriP-based plasmids in human cells

“…32,33 It has been documented that EBNA1 mediates the interaction between the FR and the DS element of OriP. [15][16][17] One possible explanation is that the plasmid segregation function of EBNA1 could be affected through a compromised linkage of the amino terminus to the carboxyl terminus of EBNA1 due to a reduction in the GlyAla domain. The experiments which located the DNA linking domains on EBNA1 were performed using GlyAla deletion mutants, and no comparative analysis of the linking function (or efficiency) of normal versus truncated EBNA1 proteins in long-term plasmid maintenance assays has yet been reported.…”

“…EBNA1 dimers bound to oriP physically link FR to the OBR (DS), resulting in the formation of a loop consisting of the intervening DNA separating these two elements. [15][16][17] This linking activity is required for the three activities of oriP mentioned above. 11 The EBNA1 protein has been extensively studied through detailed mutational analysis and several functional domains of EBNA1 have been mapped ( Figure 1).…”

Mapping the Charged Residues in the Second Immunoglobulin-like Domain of the Vascular Endothelial Growth Factor/Placenta Growth Factor Receptor Flt-1 Required for Binding and Structural Stability

“…A plasmid vector, pHEBO23 (28), containing cDNA encoding the signal peptide and first three Ig-like domains of Flt-1 fused to the human heavy chain IgG␥1 (23) was used for mutagenesis and expression. Variants were screened and verified by sequence analysis using the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer) followed by application of the Sequencher 3.01 program (Gene Codes Corp.).…”