Volume 9, Issue 3, Pages 493-503 (March 2002) Telomeric Proteins Regulate Episomal Maintenance of Epstein-Barr Virus Origin of Plasmid Replication  Zhong Deng, Larissa Lezina, Chi-Ju Chen, Svetlana Shtivelband, Wingkan So, Paul M. Lieberman  Molecular Cell  Volume 9, Issue 3, Pages 493-503 (March 2002) DOI: 10.1016/S1097-2765(02)00476-8 Copyright © 2002 Cell Press Terms and Conditions

Figure 1 Identification of EBNA1-Dependent DS Binding Proteins (A) Schematic of EBV OriP structure consisting of the family of repeats (FR) and dyad symmetry (DS) regions. (B) SL1 (HA-EBNA1+) nuclear extracts were subjected to two sequential rounds of DS or ZRE control DNA affinity purification and assayed by Western blotting with anti-HA. Second round affinity-purified proteins were eluted with 300 mM KCl (lanes 2 and 3) followed by 1000 mM KCl (lanes 4 and 5). 0.5% of the input material is shown in lane 1. EBNA1 is indicated by the arrow. (C) DS or ZRE affinity-purified proteins shown in (B) were assayed by silver staining. (D) SL1 or HeLa nuclear extracts were purified using two sequential rounds of DS DNA affinity purification. EBNA1 protein was identified by Western blotting with anti-HA. (E) Second round DS affinity-purified proteins from HeLa or SL1 cells were eluted with 300 mM KCl (lanes 1 and 2) followed by 1000 mM KCl (lanes 3 and 4) and analyzed by silver staining. (F) Polypeptides isolated by DNA affinity purification were identified by μLC/MS/MS. Molecular Cell 2002 9, 493-503DOI: (10.1016/S1097-2765(02)00476-8) Copyright © 2002 Cell Press Terms and Conditions

Figure 2 TRF2 and Tankyrase Bind to DS In Vitro and In Vivo (A) HeLa or SL1 nuclear extracts were subjected to a single round of DS-DNA affinity purification with washing at 150 or 300 mM KCl, as indicated above each lane, and assayed by Western blot for Tankyrase, TRF2, PARP, or EBNA1 as indicated. 0.5% of input protein is indicated in lanes 1 and 2. (B) SL1 nuclear extract was purified over a single round of DS (Dyad) or control (ZRE) DNA affinity chromatography and assayed by Western blotting with indicated antibodies. (C) Chromatin immunoprecipitation assay was used to measure the association of TRF2 and Tankyrase (TANK) with OriP in vivo using formaldehyde crosslinked Raji cells. Input or immunoprecipitated DNA (3-fold dilutions) was amplified with primers specific for EBV OriP or BRLF1 sequences. Chromatin was precipitated with antibodies to TRF2, Tankyrase, EBNA1, or control antibody FLAG-M2 (Sigma). Quantification of each PCR product is shown below. Molecular Cell 2002 9, 493-503DOI: (10.1016/S1097-2765(02)00476-8) Copyright © 2002 Cell Press Terms and Conditions

Figure 3 Cooperative Binding of TRF2 and EBNA1 to DS (A) Schematic of EBNA1 binding sites and nonamer repeats in DS and mutated nonamer repeats in DSΔa,b,c. (B) Coomassie brilliant blue staining of SDS-PAGE gels showing affinity-purified TRF2 and EBNA1 proteins (100 ng) derived from baculovirus-infected Sf9 cells. (C) Increasing concentrations of TRF2 (12.5–50 ng) were assayed for EMSA binding to DS (lanes 1–8) or DSΔa,b,c (lanes 9–16) in the absence or presence of 5 ng of EBNA1 (as indicated above). (D) Quantification of DNA binding shown in (C). (E) Increasing concentrations of EBNA1 (2.5–10 ng) were assayed for EMSA binding to DS (lanes 1–8) or DSΔa,b,c (lanes 9–16) in the absence or presence of 25 ng TRF2. (F) Quantification of DNA binding shown in (E). Molecular Cell 2002 9, 493-503DOI: (10.1016/S1097-2765(02)00476-8) Copyright © 2002 Cell Press Terms and Conditions

Figure 4 EBNA1 Stimulates TRF2 Binding to the DS Nonamer Repeats Recombinant EBNA1 and TRF2 generated from baculovirus were assayed by DNase I footprinting of the OriP DS region. “G” and “G+A” are chemical sequencing ladders. EBNA1 binding sites are indicated as sites 1–4. Nonamer repeats (TTAGGGTT) are indicated “a,” “b,” and “c.” (A and B) TRF2 (100 ng) and EBNA1 (50 ng) were compared alone or together for DNase footprinting on the noncoding (A) or coding (B) strand. (C) Wild-type (wt) and nonamer substitution mutant (Δa,b,c) were used as probes for DNase I footprinting for TRF2 over a concentration range of 12–100 ng (3-fold dilutions) in the absence or presence of EBNA1 (50 ng). (D) Sequences protected by EBNA1 and TRF2 in DNase I footprinting assays. Molecular Cell 2002 9, 493-503DOI: (10.1016/S1097-2765(02)00476-8) Copyright © 2002 Cell Press Terms and Conditions

Figure 5 TRF1 and TRF2 Affect OriP-Dependent DNA Replication Function (A) OriP-containing pHEBO plasmid was assayed for transient DNA replication in transfected 293 cells. pHEBO was cotransfected with pHA-EBNA1 (lanes 3–6) or with control vector (lane 2) and with either TRF1 (lane 4), ΔTRF2 (lane 5), TRF2 (lane 6), or control expression vector CMV2-FLAG (lane 3). DpnI-resistant DNA linearized with BamHI is indicated for lanes 2–6 (top panel). The same DNA samples were restricted with BamHI only (lower panel). BamHI-digested pHEBO (0.5 ng) was used as a reference in lane 1. pHEBO-specific probes were generated by random priming for Southern blots. HA-EBNA1 expression levels in transfected cells were assayed by Western blotting with anti-HA. Flag-TRF1 and Flag-ΔTRF2 were assayed by Western blotting with anti-Flag. TRF2 and ΔTRF2 were detected by anti-TRF2 antibody. DpnI-resistant pHEBO DNA from three similar experiments was quantified by PhosphorImager analysis. (B) DS wt- or DSΔa,b,c-containing plasmids were assayed for transient DNA replication using DpnI/BamHI digestion (top panel). BamHI-digested DNA is shown in the lower panel. Cells were transfected with expression vectors for EBNA1 (lanes 2–4 and 6–8), TRF1 (lanes 3 and 7), ΔTRF2 (lanes 4 and 8), or CMV2-FLAG control vector (lanes 1, 2, 5, and 6). Quantification of DpnI-resistant DNA by PhosphoImager analysis is shown in the bar graph below. Molecular Cell 2002 9, 493-503DOI: (10.1016/S1097-2765(02)00476-8) Copyright © 2002 Cell Press Terms and Conditions

Figure 6 PAR-Dependent Regulation of OriP Plasmid Maintenance (A) Akata cells electroporated with OriP wt or OriPΔa,b,c were selected by FACS for equal numbers of GFP-positive cells, cultured identically for 3 weeks, and assayed by Southern blot for maintenance of OriP plasmids. (B) D98/HR1 cells transfected with OriP were selected by FACS for GFP-positive cells and cultured with 10 mM niacinamide, 50 μM hydroxyurea, or no treatment for 3 weeks and then assayed for plasmid maintenance by Southern blot. (C) Same as in (B), except cells were subjected to a second cell sorting after 3 weeks in culture. Equal numbers of GFP-positive cells were isolated immediately prior to Southern blotting analysis. (D) Nuclear extracts derived from D98 cells treated with hydroxyurea, niacinamide, or no treatment were subjected to DS-DNA affinity purification and Western blotting with antibody specific for PAR modification (α-PAR) or EBNA1 protein (lower panel; α-EBNA1). (E) DS affinity-purified proteins from HeLa or SL1 nuclear extracts were incubated with 32P-NAD and analyzed by autoradiography of SDS-PAGE gels. (F) DS affinity-purified proteins from SL1 cells were subjected to incubation with (+) or without (−) 1 mM NAD and assayed by Western blotting with antibodies specific for Tankyrase (top panel), EBNA1 (middle panel), or TRF2 (lower panel). Molecular Cell 2002 9, 493-503DOI: (10.1016/S1097-2765(02)00476-8) Copyright © 2002 Cell Press Terms and Conditions