ORC binding to TRF2 stimulates OriP replication

Abstract

In higher eukaryotes, the origin recognition complex (ORC) lacks sequence-specific DNA binding, and it remains unclear what other factors specify an origin of DNA replication. The Epstein-Barr virus origin of plasmid replication (OriP) recruits ORC, but the precise mechanism of ORC recruitment and origin activation is not clear. We now show that ORC is recruited selectively to the dyad symmetry (DS) region of OriP as a consequence of direct interactions with telomere repeat factor 2 (TRF2) and ORC1. TRF-binding sites within DS stimulate replication initiation and facilitate ORC recruitment in vitro and in vivo. TRF2, but not TRF1 or hRap1, recruits ORC from nuclear extracts. The amino-terminal domain of TRF2 associated with a specific region of ORC1 and was necessary for stimulation of DNA replication. These results support a model in which TRF2 stimulates OriP replication activity by direct binding with ORC subunits.

Figure 1

Telomere repeats recruit ORC in vitro. (A) Schematic of EBV DNA fragments and telomere repeat substitution mutations used for DNA affinity. (B) DNA affinity assay with FR, DS or Qp template DNA and Raji nuclear extracts. Bound proteins were monitored by western blotting with antibodies to ORC2, TRF2 or EBNA1. (C) DNA affinity with control BKS, DS nm⁻ or DS wt DNA and Raji nuclear extracts was analysed with antibodies to EBNA1, TRF2, hRap1, TRF1, ORC2 and PCNA. (D) DNA affinity as in (C) with templates for control BKS, DS (1+2) nm⁻ or DS (1+2). EBV, Epstein–Barr virus; EBNA1, EBV nuclear antigen 1; DS, dyad symmetry; FR, family of repeats; ORC, origin recognition complex; OriP, origin of plasmid replication; PCNA, proliferating-cell nuclear antigen; TRF, telomere repeat factor; wt, wild type.

Figure 2

Telomere repeats function in OriP replication and ORC recruitment in vivo. (A) DNA replication assay for DS (1+2) or DS (1+2) nm⁻ in the absence (−) or presence (+) of EBNA1 transfected into HeLa cells. (B) ChIP assay of EBNA1, TRF2, ORC2, MCM3 or control IgG at DS wt or DS nm⁻ in transfected D98/HR1 cells with primers specific for the plasmid-based DS region. (C) ChIP assay of EBNA1, TRF2, ORC2, MCM3 or control IgG binding to DS in EBV-positive ZKO-293 cells transfected with shTRF2, shEBNA1 or control plasmids. ChIP, chromatin immunoprecipitation; DS, dyad symmetry; EBNA1, Epstein–Barr virus nuclear antigen 1; MCM, minichromosome maintenance; ORC2, origin recognition complex 2; OriP, origin of plasmid replication; TRF2, telomere repeat factor 2; wt, wild type.

Figure 3

ORC interacts with TRF2. (A) Coomassie stain of purified GST, GST–hRap1, GST–TRF2 and GST–TRF1. (B) Western blot with ORC2 antibodies of GST pull-down assays using Raji nuclear extract and various GST proteins shown in (A). Ethidium bromide (EtBr; 100 μg/ml) was included in the binding reactions, as indicated. (C) Co-immunoprecipitation with antibodies to EBNA1, TRF2 or control IgG. Immunoprecipitates were assayed by western blot with antibodies to EBNA1, TRF2, ORC2 or control PCNA. (D) Co-immunoprecipitation assay with anti-TRF2 and western blot with anti-ORC2 in Raji (top) or HeLa (bottom) nuclear extracts. (E) FLAG–ORC2 was purified from stable cell lines and assayed for direct interaction with purified GST or GST–TRF2 by western blot with anti-FLAG. EBNA1, Epstein–Barr virus nuclear antigen 1; GST, glutathione S-transferase; IB, immunoblotting; IP, immunoprecipitation; ORC, origin recognition complex; PCNA, proliferating-cell nuclear antigen; TRF, telomere repeat factor.

Figure 4

The basic region of TRF2 interacts with ORC and is required for OriP replication function. (A) Truncation mutants of TRF2 were fused to GST and assayed for ORC2 recruitment with Raji nuclear extracts. Bound ORC2 was identified by western blot with anti-ORC2. (B) Coomassie stain of GST–TRF2 truncation mutants, as indicated in (A). (C) Summary of ORC2 binding to GST–TRF2 truncation mutants. (D) DNA replication assay of OriP in ZKO-293 cells co-transfected with FLAG-tagged TRF2, TRF2ΔB, TRF2ΔBΔM or vector control. (E) Protein expression was confirmed by western blotting of whole-cell extracts derived from transfected ZKO-293 cells used in replication assay of (D) and probed with anti-FLAG, anti-EBNA1 or anti-PCNA loading control. EBNA1, Epstein–Barr virus nuclear antigen 1; GST, glutathione S-transferase; IB, immunoblotting; ORC, origin recognition complex; OriP, origin of plasmid replication; PCNA, proliferating-cell nuclear antigen; TRF2, telomere repeat factor 2.

Figure 5

TRF2 basic region binds directly to a subdomain of ORC1. (A) Diagram of the known functional domains of ORC1, including the BAH (aa 1–201), the ATPase (529–782) and the AIF-C1 interacting domain (210–239). (B) Coomassie stain of GST, and GST–ORC1 amino acids (1–200), (512–862) and (201–511). (C) HeLa cell nuclear extracts were incubated with GST or GST–ORC1 fragments as indicated, and analysed by western blot with an antibody specific for TRF2. Input represents 5% of the HeLa nuclear extract used for GST interaction assays. (D) DNA replication assay for OriP in ZKO-293 cells co-transfected with CMV–FLAG vector control or FLAG–ORC1 (aa 201–511). (E) Western blot with antibodies to FLAG–ORC1 (aa 201–511), EBNA1 or control PCNA corresponding to replication assay shown in (D). EBNA1, Epstein–Barr virus nuclear antigen 1; GST, glutathione S-transferase; ORC, origin recognition complex; OriP, origin of plasmid replication; PCNA, proliferating-cell nuclear antigen; TRF2, telomere repeat factor 2.