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Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication - PubMed

  • ️Mon Jan 01 2001

Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication

T M Avolio-Hunter et al. Nucleic Acids Res. 2001.

Abstract

The EBNA1 protein of Epstein-Barr virus (EBV) activates latent-phase DNA replication by an unknown mechanism that involves binding to four recognition sites in the dyad symmetry (DS) element of the viral latent origin of DNA replication. Since EBV episomes are assembled into nucleosomes, we have examined the ability of Epstein-Barr virus nuclear antigen 1 (EBNA1) to interact with the DS element when it is assembled into a nucleosome core particle. EBNA1 bound to its recognition sites within this nucleosome, forming a ternary complex, and displaced the histone octamer upon competitor DNA challenge. The DNA binding and dimerization region of EBNA1 was sufficient for nucleosome binding and destabilization. Although EBNA1 was able to bind to nucleosomes containing two recognition sites from the DS element positioned at the edge of the nucleosome, nucleosome destabilization was only observed when all four sites of the DS element were present. Our results indicate that the presence of a nucleosome at the viral origin will not prevent EBNA1 binding to its recognition sites. In addition, since four EBNA1 recognition sites are required for both nucleosome destabilization and efficient origin activation, our findings also suggest that nucleosome destabilization by EBNA1 is important for origin activation.

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Figures

Figure 1
Figure 1

The DNA fragments used to reconstitute nucleosomes. A schematic representation of the DNA fragments assembled into nucleosome core particles showing the positioning of EBNA1 binding sites 1, 2, 3 and 4. (A) The DS DNA fragment showing the location of the 118 bp DS element (DS). (B) Centered site 1+2 DNA fragment. (C) Site 1+2 DNA fragment with the same site positioning as in the DS DNA fragment.

Figure 2
Figure 2

EBNA452–641 forms a ternary complex with the DS-nucleosome. (A) EBNA452–641 was titrated with DS-nucleosomes (Nuc) or with naked DS DNA fragments (DNA), and complexes were analyzed by EMSAs. The intermediates formed during the filling of the four EBNA1 sites in the naked DS DNA are labeled according to the number of sites bound (1–4). (B) DS-nucleosomes (Nuc) or ternary complexes formed between EBNA452–641 and DS-nucleosomes (Nuc+EBNA1) were challenged by the addition of increasing amounts of unlabeled DS DNA competitor prior to acrylamide gel electrophoresis. DS competitor added is shown as fold excess over the labeled DS DNA fragments.

Figure 3
Figure 3

DNase I footprints of ternary complexes. Nucleosomes containing DS DNA fragments (Nuc) were incubated with increasing amounts of EBNA452–641 to form ternary complexes, then subjected to DNase I footprint analysis. Bands protected by EBNA452–641 binding are indicated (arrows and brackets) as are DNase I-hypersensitive bands induced by EBNA452–641 (*). The DNase I digestion patterns of the naked DS DNA fragment (lane 2) and EBNA452–641 bound to the naked DS (lane 1) are also shown. The positions of EBNA1 binding sites 1–4 are indicated. (A) and (B) are footprints of opposite DNA strands.

Figure 4
Figure 4

Functional EBNA1 forms a ternary complex with the DS-nucleosome. The DS-nucleosome (Nuc) was incubated with sufficient EBNA1 (7 pmol) to form a ternary complex (lane 4) then this complex was challenged by the addition of a 1000-fold excess of unlabeled DS DNA (DS Comp; lane 5). Complexes were analyzed by EMSA. The positions of the labeled DS DNA (lane 1), the DS-nucleosome (lane 2) and EBNA1 bound to naked DS DNA (lane 6) are also shown. Lanes 1 and 6 contain labeled DS DNA and lanes 2–5 contain the labeled DS-nucleosome.

Figure 5
Figure 5

Histones are displaced from ternary complexes upon non-specific competitor DNA challenge. Nucleosomes containing DS DNA fragments were incubated with 12 pmol of EBNA452–641 to form ternary complexes (Nuc+EBNA1) then were challenged by the addition of increasing amounts of plasmid competitor DNA. Nucleosomes that had not been incubated with EBNA1 (Nuc) were also challenged with plasmid competitor. The positions of the naked DS DNA (DNA) and DS DNA bound by EBNA452–641 (DNA+EBNA1) are indicated. (A) Nucleosomes were formed from purified chicken histone octamers by salt dialysis. (B) Nucleosomes were formed from HeLa oligonucleosomes by octamer transfer.

Figure 6
Figure 6

The DNA binding and dimerization domain of EBNA1 is sufficient for nucleosome disruption. EBNA459–607 (15 pmol) was incubated with a DS-nucleosomes (lane 2) to form a ternary complex (lane 3), which was challenged by the addition of plasmid competitor DNA (lane 4). The positions of naked DS DNA fragments, either unbound (lane 1) or bound by EBNA459–607 (lane 5) are also indicated.

Figure 7
Figure 7

Two EBNA1 recognition sites are insufficient for nucleosome destabilization. (A) A DNA fragment containing sites 1 and 2 with the same positioning as in the DS DNA fragment (Fig. 1C) was assembled into a nucleosome (lane 2) then titrated with EBNA452–641 to form a ternary complex (lanes 3–7; Nuc+EBNA1). EBNA452–641 bound to the naked DNA fragment (lanes 8 and 9) is shown and positions of shifted complexes representing EBNA452–641 binding to one or two of these sites is indicated. (B) Ternary complexes from (A) (lane 4) were challenged with 500 ng of plasmid competitor DNA (lane 5), as were nucleosomes that lacked EBNA452–641 (lane 3). The positions of the naked DNA fragments, unbound (DNA) or bound by EBNA452–641 (DNA+EBNA1) are shown. Note that while two bands are observed in the vicinity of the DNA+EBNA1 arrow after addition of EBNA452–641 to the nucleosomes (lanes 4 and 5), only the lower band corresponds to EBNA452–641 bound to naked DNA. The composition of the upper band is not known.

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