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

Abstract

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.

FIG. 1

EBNA1 binding sites at DS of oriP and mutations that alter their spacing. (A) EBNA1 dimers (double spheres) bound to their four sites at the ∼120-bp replicator DS. Distances between the centers of the binding sites and some restriction sites are indicated. (B) Nucleotide sequences between the active pairs of EBNA1 sites 3 and 4 and 1 and 2 for wild-type (WT) and mutants, with the outermost two nucleotides of each EBNA1 site underlined. The boundaries of the 16-bp palindromic EBNA1 binding site were determined in a study of synthetic sites and from crystallographic data (3, 5). Substituted or inserted nucleotides are in bold; dots indicate deleted nucleotides.

FIG. 2

EBNA1 binding sites at DS must be exactly 21 bp apart, center to center, to support plasmid replication. (A) At 60 h after transfection of EBNA1-positive 143B cells, plasmids were isolated, digested with BamHI to linearize them and with DpnI to test for replication (loss of bacterial adenine methylation), and detected by Southern analysis. Arrow, full-length, DpnI-resistant plasmid. Plasmids carried oriP with DS intact (WT), with only EBNA1 sites 2 and 4 active (2&4), with only EBNA1 sites 3 and 4 present (3&4), or with 3&4 with altered spacing (−1, −2, and +2). 3&4c and its derivatives, −2c and +2c, also contain the T-to-A consensus mutation at nt 9046 of site 4 (38). The average signals for the replicated plasmid relative to that for the wild type are shown above the blot image. Duplicate transfections were analyzed except for −1 (nt 9050) in lane 10. For lane 1, 4 ng of plasmid was mixed with DNA from nontransfected cells and treated similarly, as a control for DpnI digestion. (B) Test for maintenance of the same set of plasmids in cells grown under selection for 3 to 4 weeks. The Southern analysis of uncut plasmids extracted from 5 × 10⁶ cells is shown. In the last two lanes, 50 and 250 pg of pHEBo was loaded, corresponding to 1.5 and 15 copies per cell. The supercoiled (S) and relaxed circular (RC) forms of the plasmid are indicated at the left. (C) Assay for replication of plasmids containing only EBNA1 sites 1 and 2 at DS during 50 h following transfection, as for panel A, but with only the most relevant part of the blot image shown. 1&2c contains consensus mutations in sites 1 and 2, as does the +1 mutant, for which duplicate transfections of two different plasmid preparations are shown (lanes 7 to 10). 1&2m has 1 bp deleted from the center of site 2. (D) Mutations at DS do not affect the inefficient replication of plasmids that can be detected in the absence of EBNA1. At 47 h after transfection of 293 cells, plasmids were extracted and analyzed as for panel A. Forty-five percent of the DNA was digested with BamHI and DpnI for the upper blot; for the lower blot, 5% of each was digested with BamHI to measure DNA uptake by cells. The average replication of each relative to that of pHEBo (WT), normalized for DNA uptake, is indicated above the upper image. The vector lacking oriP was tested for lanes 1 and 2.

FIG. 3

Binding of EBNA1 to sites 3 and 4 with normal or altered spacing. wt, normal spacing between sites 3 and 4; the changes in spacing are indicated by −2 to +10 (base pairs). (A) Electrophoresis of 15 fmol of end-labeled DNAs, with the indicated spacing between sites 3 and 4, through a 4% polyacrylamide gel after binding by EBNA1 NΔ407 in a limiting amount (30 fmol of dimer; lanes 2 to 8) or in excess (60 fmol of dimer; lanes 9 to 15) or without EBNA1 (lane 1). The DNAs spanned DS but had EBNA1 sites 1 and 2 deleted (lanes 2 to 5 and 9 to 12) or inactivated by point mutations (lanes 6 to 8 and 13 to 15). In both cases, the DNAs with wild-type spacing were 277 and 278 bp long, respectively, with the center 11 bp to the right of site 3. At the left, the positions of free DNA (F) and the complexes with one site or two sites bound are indicated. (B) Helical phase analysis of DNA bending at sites 3 and 4. Shown are the mobilities of the complexes relative to that of free DNA, measured using the gel shown in panel A and a similar one. The mean values of three determinations (six for wild-type spacing) are indicated; brackets, range of measurement.

FIG. 4

Circular permutation assay for EBNA1-induced DNA bending. Circularly permuted 288-bp DNAs containing the left half of DS were generated from the construct shown in panel B. EBNA1 sites 3 and 4 are indicated. (A) Internally labeled DNA (15 fmol) was excised at the restriction sites indicated, mixed with 60 fmol of EBNA1 NΔ407, and electrophoresed through a 4% polyacrylamide gel. The positions of free DNA (F) and the complexes with one site or two sites bound are indicated at the left. For lanes 1 to 6, the DNA was excised with XbaI, MwoI, ApaI, BstYI, Bsu36I, and TaqI, respectively. (C) Mobilities of the complexes relative to that of unbound DNA, measured from two independent gels and plotted against the position of the end of each DNA within the nonpermuted sequence.

FIG. 5

EMSA analysis of five types of complexes that result from binding of limiting amounts of EBNA1 to its four sites at DS. The complexes are labeled 1, 2 IP, 2 OP, 3, and 4 based on the number of EBNA1 binding sites that are bound by EBNA1 in each complex. IP and OP are explained in the text. (A) Effects of eliminating each EBNA1 site individually. The substitution mutations, 1-Bst, 2-Bst, etc., eliminate binding to each site (38). In lanes 3 to 6, the EBNA1 sites that are bound in the predominant 2 OP complex are indicated in each lane. At the right, the two EBNA1 sites that are occupied in each 2 IP complex are indicated. F, free DNA. (B) Effects of eliminating binding to different pairs of sites. The DNAs that were tested each contained double point mutations (dpm) in two different binding sites, as constructed by Harrsion et al. (16). For example, sites 2 and 3 are both inactive with dpm2 + 3. Lanes 6 to 10 are a repetition of lanes 1 to 5 except that the ratio of EBNA1 dimers to binding sites is doubled. (C) Effects of increasing the distance between sites 2 and 3 by 5 or 10 bp using the oriP mutants in2/3[5] and in2/3[10], respectively, described by Harrison et al. (16). (A to C) Fifteen femtomoles of each DNA was used with the following amounts of EBNA1 NΔ407 dimer: A and C, none for lane 1, 60 fmol otherwise; B, 30 fmol for lanes 1 to 4, 60 fmol for lanes 5 to 9, 120 fmol for lane 10, none for lane 11.