We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.
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 EpsteinBarr 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 aminoterminal 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.
Epstein-Barr virus OriP confers cell cycle-dependent DNA replication and stable maintenance on plasmids in EBNA1-positive cells. The dyad symmetry region of OriP contains four EBNA1 binding sites that are punctuated by 9-bp repeats referred to as nonamers. Previous work has shown that the nonamers bind to cellular factors associated with human telomeres and contribute to episomal maintenance of OriP. In this work, we show that substitution mutation of all three nonamer sites reduces both DNA replication and plasmid maintenance of OriP-containing plasmids by 2.5-to 5-fold. The nonamers were required for high-affinity binding of TRF1, TRF2, and hRap1 to the dyad symmetry element but were not essential for the binding of EBNA1 as determined by DNA affinity purification from nuclear extracts. Chromatin immunoprecipitation assays indicated that TRF1, TRF2, and hRap1 bound OriP in vivo. Cell cycle studies indicate that TRF2 binding to OriP peaks in G 1 /S while TRF1 binding peaks in G 2 /M. OriP replication was inhibited by transfection of full-length TRF1 but not by deletion mutants lacking the myb DNA binding domain. In contrast, OriP replication was not affected by transfection of full-length TRF2 or hRap1 but was potently inhibited by dominant-negative TRF2 or hRap1 amino-terminal truncation mutants. Knockdown experiments with short interfering RNAs (siRNAs) directed against TRF2 and hRap1 severely reduced OriP replication, while TRF1 siRNA had a modest stimulatory effect on OriP replication. These results indicate that TRF2 and hRap1 promote, while TRF1 antagonizes, OriP-dependent DNA replication and suggest that these telomeric factors contribute to the establishment of replication competence at OriP.Epstein-Barr virus (EBV) is a lymphotropic gammaherpesvirus that can be cultured in latently infected B-cell lines as a multicopy extrachromosomal plasmid (reviewed in reference 27). The latent viral genome can be isolated from a variety of tumor tissues and is causally linked with Burkitt's lymphoma, nasopharyngeal carcinoma, and lymphoproliferative disorders in the immunosuppressed population (reviewed in reference 52). In most, if not all, latent infections the virus-encoded EBV nuclear antigen 1 (EBNA1) can be detected. Genetic and biochemical experiments have established that EBNA1 is essential for the maintenance of the viral genome during latency (34, 74). EBNA1 binds with high avidity to three regions of the viral genome, namely, the family of 30-bp repeats (FR), the dyad symmetry (DS) region, and the Q promoter (49). The FR and DS together comprise the viral origin of plasmid replication (OriP). The DS is required for DNA replication initiation, while FR stimulates DS replication and provides plasmid maintenance activity (3,11,29,55,73). The Q promoter has no known replication activity but is thought to autoregulate EBNA1 RNA transcription (62). EBNA1 binding to OriP is sufficient to confer plasmid replication and maintenance in most human and primate cell types but may be restricted in murine and hamster c...
The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA. In total it mimics 22 phosphate groups over approximately 24 bp of DNA. This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes. We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI. One dimer binds to the core methyltransferase and two to the complete bifunctional restriction and modification enzyme. Ocr can also bind to the component subunits of EcoKI. Binding affinity to the methyltransferase core is extremely strong with a large favourable enthalpy change and an unfavourable entropy change. This strong interaction prevents the dissociation of the methyltransferase which occurs upon dilution of the enzyme. This stabilisation arises because the interaction appears to involve virtually the entire surface area of ocr and leads to the enzyme completely wrapping around ocr.
Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knockdown of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.Epstein-Barr virus (EBV) is a human herpesvirus that establishes lifelong latent infections causally linked to several cancers, including Burkitt's lymphoma and nasopharyngeal carcinoma (23, 32). The latent form of EBV exists as a multicopy extrachromosomal plasmid that replicates semiconservatively during the S phase of the cell cycle (1, 50). Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein required for stable episomal maintenance of the viral genome (21, 26, 51) (reviewed in references 27 and 43). EBNA1 binds to two regions of the viral origin of plasmid replication (OriP), referred to as the family of repeats (FR) and the dyad symmetry (DS) element (31). FR is essential for plasmid maintenance, while DS is required for initiation of OriP-dependent DNA replication (18, 52). Cellular factors, like the origin recognition complex (ORC) and minichromosome maintenance (MCM) proteins, which regulate chromosomal DNA replication and cell cycle licensing, have been shown to associate with the DS region of OriP (9,16,35). In addition to these replication factors, several telomere-associated proteins, including telomere repeat factors (TRF1 and TRF2) and hRap1, interact with three nonamer repeats (TTAGGGTTA) that are interspersed with EBNA1 binding sites in the DS (13,14).In a previous study, we had shown that TRF2 and hRap1 facilitate DS-dependent DNA replication and OriP plasmid maintenance (13). In addition to TRF1, TRF2, and hRap1, we had also found that the telomere-associated poly-ADP ribose polymerase tankyrase 1 (TNKS1) was associated with the DS, using DNA affinity chromatography and chromatin immunoprecipitation assays (14). TNKS1 was originally identified as a TRF1-interactin...
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