Multiple regions within EBNA1 can link DNAs

Abstract

Epstein-Barr virus nuclear antigen 1 (EBNA1) can bind specifically to two clusters of sites within the Epstein-Barr virus plasmid origin of DNA replication (oriP). EBNA1 activates DNA replication mediated by oriP and can also activate transcription and retain DNA in cells when bound site specifically. EBNA1 bound to oriP physically links the two clusters of EBNA1-binding sites, resulting in loop formation by the intervening DNA. To elucidate the contribution of DNA linking by EBNA1 to its biological activities, we identified regions within it that can independently link DNAs to which they are bound. An electrophoretic mobility shift assay was used to detect this activity. Proteins which link DNA aggregate that DNA into large lattices. Proteins which cannot link DNA but still bind to DNA retard the mobility of that DNA but do not cause it to form lattices. Amino-terminal truncations were used to map the amino-terminal limit of a minimal DNA-linking domain approximately to amino acid 372 of EBNA1. To map the carboxy-terminal limit of this minimal domain, fusion proteins containing the DNA-binding domain of GAL4 and fragments of EBNA1 were generated and studied. This approach identified the carboxy-terminal limit of this minimal domain to be approximately amino acid 391 and verified its amino-terminal limit. Internal deletions within a truncated EBNA1 derivative verified the importance of this region. Two additional fragments of EBNA1, each of which independently conferred DNA-linking activity on the domain of GAL4 which binds DNA, were identified within amino acids 54 to 89 and amino acids 331 to 361. Therefore, EBNA1 contains at least three regions that can act independently to link DNAs and that may act in concert within intact EBNA1.

The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element

“…The biochemical properties of EBNA-1 do, however, combine to provide a model for this maintenance function. EBNA-1 binds DNA site-specifically (Rawlins et al, 1985;Ambinder et al, 1990Ambinder et al, , 1991Bochkarev et al, 1996) and links bound DNAs through proteinprotein associations mediated by its multiple 'linking' domains (Laine and Frappier, 1995;Mackey et al, 1995;Mackey and Sugden, 1997). On binding site-specifically, both to FR and to hypothetical sites on host chromosomes, EBNA-1 molecules can link EBV or oriP plasmids to chromatin to ensure their retention in daughter nuclei at the end of mitosis.…”

The Minimal Replicator of Epstein-Barr VirusoriP

“…The 278-bp DNAs were centered between nucleotides 9081 and 9082, located between EBNA-1 sites 2 and 3. Then, 15 fmol of endlabeled DS DNA (adjusted to 2,000 dpm per fmol using unlabeled DNA), 2 g of salmon sperm DNA, 60 or 240 fmol of EBNA-1 N⌬407 dimers (26), and 50 g of protein from COS7 cells were combined in a volume of 15 l containing 128 mM KCl, 6 mM (NH 4 ) 2 SO 4 , 5 mM MgCl 2 , 1.3 mM dithiothreitol, 10 mM Tris-HCl (pH 7.4), and 7.6% glycerol. After 10 min at room temperature, the samples were analyzed by electrophoresis through a 4% polyacrylamide gel as described earlier (6) except that 50 mM Tris-HCl-190 mM glycine (pH 8.5) was used as the electrophoresis buffer.…”

“…After 10 min at room temperature, the samples were analyzed by electrophoresis through a 4% polyacrylamide gel as described earlier (6) except that 50 mM Tris-HCl-190 mM glycine (pH 8.5) was used as the electrophoresis buffer. Purified N⌬407 protein, produced in Escherichia coli, was a generous gift of D. Mackey and B. Sugden (26). The COS7 protein, a ribosome-free soluble fraction (S100) of a whole-cell extract made by using 0.5 M KCl, had no effect on the number of complexes seen or their mobilities but had a "carrier" effect, reducing by severalfold the amount of EBNA-1 required.…”

“…4. To allow resolution of individual bands in the assay, we used a truncated EBNA-1 protein, N⌬407, which lacks the linking domains of the amino-terminal half of the protein (26). Duplicate plates of 293 cells were transfected with the oriP test plasmids as indicated.…”

Functional Analyses of the EBNA1 Origin DNA Binding Protein of Epstein-Barr Virus

“…Two Gly-Arg-rich regions are present between residues 40 and 55 and residues 325 and 376. The latter region corresponds to the DNA looping or linking domain which has been shown to mediate homotypic interactions at a distance between DNA-bound EBNA1 molecules (5,16,24,28), as well as heterotypic interactions with at least two cellular proteins (42,48). This region has also been reported to bind RNA (43).…”