- Email: [email protected]
The Origin Recognition Complex Localizes to Telomere Repeats and Prevents Telomere-Circle Formation
Current Biology 17, 1989–1995, November 20, 2007 ª2007 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2007.10.054
Report The Origin Recognition Complex Localizes to Telomere Repeats and Prevents Telomere-Circle Formation Zhong Deng,1 Jayaraju Dheekollu,1 Dominique Broccoli,2 Anindya Dutta,3 and Paul M. Lieberman1,* 1The Wistar Institute Philadelphia, Pennsylvania 19104 2Memorial Health University Medical Center Savannah, Georgia 31404 3University of Virginia Charlottesville, Virginia 22908
Summary Chromosome ends are maintained by telomere-repeat-binding factors (TRFs) that coordinate DNA end protection with telomere replication [1, 2]. The origin recognition complex (ORC) coordinates bidirectional DNA replication at most chromosomal sites, but it is also known to function in transcriptional silencing, heterochromatin formation, and sister-chromatid cohesion [3, 4]. We now show that ORC localizes to telomere repeats and contributes to telomere maintenance. We found that ORC subunits can be affinity purified with telomere-repeat DNA along with other components of the known ‘‘shelterin’’ complex. ORC subunits colocalized with telomere-repeat foci and coimmunoprecipitated with TRF2 but not TRF2 lacking its amino-terminal basic domain (TRF2DB). ORC2 depletion or hypomorphic cell lines caused a loss of telomere-repeat signal intensity and the appearance of dysfunctional telomeres, including telomere-signalfree ends and telomere-repeat-containing double minutes. Two-dimensional agarose gel electrophoresis revealed that ORC2 depletion increased telomere circle formation, comparable to the overexpression of TRF2DB. A similar increase in telomere circle formation was induced by hydroxyurea treatment, providing evidence that replication stress produces telomere circles. These findings suggest that ORC recruitment by TRF2 contributes to telomere integrity by facilitating efficient telomere DNA replication and preventing the generation of telomere-repeat-containing circles. Results and Discussion The completion of DNA replication at the ends of linear chromosomes can be problematic not only because of the end-replication problem but also as a result of repetitive DNA and nucleoprotein-structures that might act as replication fork barriers [5, 6]. The telomere repeats and the proteins that associate with these repeats provide chromosome ends with protection from nucleolytic degradation and end-to-end fusions that would otherwise lead to genetic instability [7, 8]. Telomere-repeat length is maintained by the interplay of telomerase,
*Correspondence: [email protected]
recombination-based lengthening, and semiconservative DNA replication [9, 10]. The telomere-repeat-binding factors that protect telomeres from degradation and fusions are also important for maintaining telomere length and coordinating the access of replication proteins to telomere DNA [10, 11]. It is generally thought that most replication initiates outside of telomere repeats at chromosomal origins bound by ORC [12–14]. In lower eukaryotes, ORC is known to bind to specific sites in the subtelomere repeats. However, in higher eukaryotes ORC lacks sequence-specific DNA binding, and it is not clear how it is recruited to specific chromosomal regions. In an earlier study, we found that TRF2 facilitated ORC recruitment to a viral origin of DNA replication [15]. This earlier study raised the question of whether TRF2 recruited ORC to cellular telomeres and whether ORC plays a role in telomere maintenance. To investigate the possibility that ORC bound to telomere-repeat DNA, we isolated nuclear factors that bound to telomere repeats by using DNA affinity chromatography. Proteins from Raji cell nuclear extracts were purified with biotinylated DNA containing 12 telomere repeats (TTAGGG) or control (BKS) DNA bound to streptavidin magnetic beads. Affinity-purified proteins were visualized by colloidal blue staining and then identified by mass spectrometry (Figures 1A and 1B). The major proteins isolated were among those already known to bind directly to telomere repeats, including TRF2, hRap1, and TRF1 [1]. Other members of the telomere-associated shelterin complex, including TIN2, POT1, and TPP1, were also identified [1]. Notably, we identified four of the six ORC subunits in the telomere-repeat DNA affinity-purified samples. ORC1, ORC2, and ORC4 binding specificity for telomere repeats was verified by western blotting (Figure 1B). Heterochromatin protein 1 (HP1a) [16], which has been reported to interact with ORC [17, 18], was weakly detected by western blot, as was the recently identified telomere-associated 50 exonuclease SNM1B (also referred to as Apollo) [19, 20]. ORC2 is an essential core subunit of ORC and was further analyzed for its association with telomere repeats in vivo by chromatin immunoprecipitation (ChIP) (Figure 1C). Telomere-repeat DNA was enriched in ORC2-specific ChIP (3.6% input) but not in ChIP with control IgG (<0.4% input) or antibodies to the EBV-encoded EBNA1 protein that binds OriP. ORC2 bound telomere-repeat DNA to a greater extent than to a well-characterized viral origin of DNA replication (DS) or to cellular GAPDH DNA. The interaction of ORC2 with telomere DNA was also observed to varying extents in cells with different telomere-maintenance mechanisms, including telomerase-positive HCT and HeLa, telomerase-negative cells U2OS, which maintain long telomeres by a recombinational mechanism referred to as alternative lengthening of telomeres (ALT), and telomerase-negative primary human peripheral blood mononuclear cells (PBMCs). These findings indicate that ORC2 associates with telomere-repeat DNA in multiple cell types,
Current Biology Vol 17 No 22 1990
Figure 1. ORC Associates with Telomere-Repeat DNA (A) DNA affinity purification of Raji nuclear extract with (TTAGGG)12 or control BKS DNA. Purified proteins were visualized with colloidal blue and identified with LC/MS/MS. (B) We used western-blot analysis to confirm the identity of proteins identified by LC/MS/MS. Proteins boxed in green were considered negative controls. ORC subunits are boxed in red. (C) ChIP assays with antibodies specific to TRF2, EBNA1, and ORC2 were probed with telomere-repeat DNA (TTAGGG, left panel), EBV OriP DNA (DS, middle panel), or control GAPDH DNA (right panel) in Raji cells. A quantification of three independent ChIP dot blots is shown below. (D) ChIP analysis of ORC2 or IgG control antibodies at TTAGGG (top) or Alu (bottom) sequence in Raji, HCT, HeLA, U2OS, or peripheral blood mononuclear cells from healthy donors (PBMC). Error bars represent mean values (6SD). (E) Indirect immunofluorescence (IF) was performed with antibody to ORC2 (red) and TRF2 (green) in HCT116 (Ei–Eiv) or U2OS (Ei0 –Eiv0 ) cells. Merge of ORC2 and TRF2 was combined with DAPI (Eiii and Eiii0 ) and enlarged (Eiv and Eiv0 ). The average percentage of TRF2 foci that colocalize with ORC2 foci is indicated to the right, where n is the number of images counted. (F) Immuno-FISH was performed with antibody to ORC2 (red) and TTAGGG PNA probe (green) in HCT116 or U2OS.
independent of the telomere-lengthening mechanism (Figure 1D, top panel). The association was specific for telomere-repeat DNA because ORC2 ChIP was not enriched for Alu-repeat DNA (Figure 1D, bottom panel). A
similar enrichment of ORC at telomere repeats was observed when FLAG-ORC2 or FLAG-ORC1 was ectopically expressed in HCT116 cells (Figure S1C in the Supplemental Data available online), indicating that multiple
ORC Functions in Telomere Maintenance 1991
Figure 2. TRF2 Is Required for ORC Recruitment at Telomeres (A) siTRF2 or control siRNA was transfected into HCT116 cells and assayed at 72 hr after transfection by western blotting for TRF2, ORC2, or PCNA expression levels. (B) siControl or siTRF2-transfected HCT116 cells were analyzed by ChIP assay in duplicates with anti-TRF2, anti-ORC2, or IgG control and were probed by hybridization with TTAGGG (left panel) or Alu repeats (right panel). (C) Quantification of four independent experiments represented in (B). (D) FLAG-TRF2 (top panels) or FLAG-TRF2DB (lower panels) was transfected into HCT116 cells, immunoprecipitated, and assayed by western blotting with antibodies to FLAG (top) or ORC2 (bottom). (E) TRF2DB transfected HCT116 cells or control cells were assayed by ChIP 72 hr after transfection with antibodies to TRF2, ORC2, or control IgG and probed with telomere TTAGGG, Alu repeat, or single-copy gene 36BD4 DNA. (F) Quantification of three independent ChIP assays is shown in (E).
ORC subunits associate with telomere repeats. Also, siRNA depletion of ORC2 reduced ORC2 ChIP at telomeres, further indicating that ORC2 antibodies were specific for ORC2 at telomeres (Figure S2). We next tested whether ORC2 protein colocalized with TRF2-associated telomere foci by using indirect immunofluorescence (IF) (Figures 1E and 1F). After detergent extraction that enriches for telomere-specific foci, we found that w50% of the TRF2 foci in HCT116 and w65% of the TRF2 foci in U2OS cells colocalized with ORC2 foci (Figure 1E). To determine whether the ORC2 foci colocalized with telomere-repeat DNA, we performed immuno-FISH with anti-ORC2 antibody and a TTAGGG PNA probe (Figure 1F). We observed nearly identical results to those found with TRF2 foci, in which ORC2 colocalized with w44% or w66% of the TTAGGG foci in HCT116 or U2OS cells, respectively. This colocalization was not limited to ORC2; transfected GFP-ORC1 also colocalized with TRF2-specific telomere foci to a similar extent as ORC2, in both HCT and U2OS cells (Figure S3). Additionally, DsRed-TRF2 colocalized with w68% of the GFP-ORC1 foci in transfected cells, indicating that the colocalization was not a consequence of antibody crossreactivity (Figure S4). These findings indicate that ORC1 and ORC2 associate with approximately half
of the telomere-repeat foci in telomerase-positive and ALT-positive cell lines. Previous studies demonstrated that TRF2 can interact with ORC subunits [15] (also Figures S1A and S1B). To determine whether TRF2 was required for ORC recruitment to telomeres, we tested whether ORC associated with telomere-repeat DNA in cells in which TRF2 was depleted by siRNA (Figures 2A–2C). TRF2 was efficiently depleted in HCT116 cells transfected with siTRF2 but not in cells transfected with control siRNA (Figure 2A). siTRF2 had no detectable effect on ORC2 or PCNA protein levels in transient transfection assays 72 hr after transfection. By using ChIP assays, we found that TRF2 depletion reduced ORC2 interaction with telomererepeat DNA by w5-fold (Figures 2B and 2C). A similar result was observed by IF, during which siRNA depletion of TRF2 abrogated ORC2 colocalization with TRF1 telomere foci (Figure S5). Previous studies demonstrated that TRF2 amino-terminal domain was important for interactions with ORC [15]. These findings were further substantiated by coimmunoprecipitation experiments that show ORC2 interacts with full-length TRF2 but not with TRF2 lacking the amino-terminal basic domain (TRF2DB) (Figure 2D). We next asked whether TRF2DB overexpression disrupts ORC association
Current Biology Vol 17 No 22 1992
Figure 3. Telomere-Repeat Loss and Dysfunction in ORC2 Depleted Cells (A) HCT116 cells were harvested at day 7 after three separate transfections with control, ORC2-1, or ORC2-2 siRNAs. Telomere-repeat length and intensity was measured by restriction digest of genomic DNA with AluI/MboI and Southern hybridization with DIG-labeled (TTAGGG)6 probe (middle panel) or control Alu (right panel). Ethidium-bromide staining of total DNA digest was used as a control for DNA loading (left panel). The position of MWs (kb) is indicated to the left. (B) Western blot of ORC2 in siControl, siORC2-1-, and siORC2-2-treated HCT116 cells used in the experiments shown in Figure 3A. TRF2, PCNA, and p54nrb blots were used as loading controls. (C) Relative telomere-repeat-signal loss was quantified for at least four independent experiments (Figure S7 and data not shown). (D) Telomere-repeat length of parental HCT116 and the ORC2 hypomorphic cell line e83 was measured as above at indicated time points (left panel). The right panel shows the telomere-repeat length of HCT116 cells transfected with siControl or siORC2-specific siRNA at various days after transfection. Arrowheads indicate approximate median telomere-repeat lengths of wild-type or control siRNAtransfected cells, and brackets represent median telomere signals of ORC2 hypomorphic or depleted cells. (E) Representative telomere FISH analysis on metaphase spreads for telomere defects. siRNA-transfected cells were harvested on day 7 posttransfection. White arrows indicate common telomere aberrations. (F) Magnified view of common telomere aberrations found in e83 or ORC2 depleted cells: telomere-signal-free ends (Fi–Fiii), telomere-containing double minutes (TDM, Fiv–Fvii), and sister chromatid lacking cohesion (Fviii and Fix).
ORC Functions in Telomere Maintenance 1993
with telomere-repeat DNA (Figures 2E and 2F). TRF2DB overexpression caused a w2.5-fold decrease in ORC association with telomere-repeat DNA by ChIP assay (Figure 2E, left panel). We also found that FLAG-TRF2DB failed to colocalize with ORC2 foci, whereas FLAG-TRF2 wild-type colocalized with ORC2 in IF experiments (Figure S6). These findings support the model that the TRF2 amino-terminal domain is required for ORC recruitment to telomeres [15]. The functional consequences of TRFDB overexpression and TRF2 depletion on telomere maintenance have been characterized previously [21, 22]. In HCT116 cells, we found that TRF2DB overexpression led to a loss of telomere-repeat signal intensity by w24% in restriction fragment-length assays (Figure S7E). We reasoned that if ORC was a functionally relevant target of TRF2 amino-terminal basic domain, then ORC2 depletion should have a similar effect as overexpression of TRF2DB. Two different siRNA, siORC2-1 and siORC2-2, efficiently depleted ORC2 but not control proteins TRF2, PCNA, or p54nrb from HCT116 cells (Figure 3B and Figure S8B). siORC2-1 had no significant effect on cell-cycle profile and cell proliferation, whereas siORC22 produced a slight increase in sub-G1 cells, but cells continued to proliferate at comparable rates (Figure S8C and data not shown). Repetitive transfection with siORC2-1 and siORC2-2 caused a w21 and 27% loss, respectively, of telomere-repeat signal intensity relative to control siRNA (Figure 3A). Similar loss of telomere-repeat signals by siORC2-1 was also observed in U2OS cells (Figure S7F). To eliminate the possibility that siORC depletion was due to off-target effects, we analyzed the telomere-repeat signals in an HCT116-derived cell line, referred to as e83, that is hypomorphic for ORC2 (ORC2D/2) [23]. We found that telomere-repeat signal in e83 was weaker and was lost at an accelerated rate relative to parental HCT116 cells (Figure 3D, left panel, and Figure S8D for loading controls). This accelerated loss of telomere-repeat signal was also observed when HCT116 cells were repeatedly transfected with siORC2-1, but not with control siRNA (Figure 3B, right panel and Figure S8E for loading controls; see also Figures S7B and S7C). These data indicate that ORC2 depletion by siRNA or hypomorphic cell lines leads to loss of telomere repeat DNA. This loss of telomere repeat signal is comparable to that observed when TRF2DB is overexpressed. To better assess the effect of ORC2 on individual telomere signals, we examined metaphase chromosome telomeres by fluorescence in situ hybridization (FISH) in siRNA-depleted and hypomorphic ORC2 cells (Figures 3E–3G). We found that reduced ORC2 levels resulted in several cytological telomere aberrations, including a 4- to 6-fold increase in signal-free ends and a 6- to 12fold increase in telomere-containing double minutes (Figure 3G). Overexpression of TRF2DB is also known to increase signal-free ends [22], suggesting that ORC2 might cooperate with TRF2 in maintaining telomere repeats. Double minutes containing telomere repeats were previously found enriched in cells depleted in
ERCC1 [24], but it is not clear whether ORC2 interacts with components of the ERCC1 pathway at telomeres. Interestingly, we also observed an increase in separated sister chromatids (Figures 3Fviii and 3Fix), suggesting that mammalian ORC might function in sister-chromatid cohesion similar to that described for budding yeast [25]. Overexpression of TRF2DB is known to cause telomere-circle formation [22]. The loss of a telomere-repeat signals and the accumulation of telomere cytological defects (Figure 3) suggests that ORC depletion might also cause telomere-circle formation. To test this possibility, we analyzed telomere-repeat DNA by 2D neutral agarose gel electrophoresis and Southern-blot analysis (Figure 4). We found that siORC2-1 and siORC2-2 increased the formation of telomere-repeat-circle arcs relative to linear arcs by w6.5-fold in HCT116 cells (Figure 4A). The telomere-circle arcs comigrated with self-ligated lambda DNA (Figures S9 and S10) and were detected equally with TTAGGG and CCCTAA probes (data not shown), indicating that the arcs consist of circular, doublestranded telomere DNA (data not shown). No circle arcs were observed at Alu-repeat DNA (Figure 4A, lower panel) indicating that ORC2 depletion increased telomere-circle formation specifically (Figures S9 and S10). Telomere circles were also observed at higher frequency (w2.9 fold) in ORC2 hypomorphic cell line e83 relative to HCT parental controls (Figure 4B) and in ORC2-depleted U2OS cells (w1.5-fold), which are known to have significant background levels of telomere circles (Figure 4D). We also noted that a slower migrating form of telomeric DNA accumulated in ORC2 depleted and hypomorphic cells (indicated by the arrowheads). These slow-migrating molecules might correspond to complex DNA structures, including replication and recombination intermediates. To determine whether ORC2 depletion induced telomere circles as a consequence of incomplete replication, we assayed telomere DNA structure in cells treated with low doses of the ribonucleotide reductase inhibitor hydroxyurea (HU) (Figures 4C and 4E). We found that HU treatment induced telomere-circle formation and the appearance of slow-mobility structures, similar to ORC2 depletion. This suggests that telomere-circle formation can be caused by incomplete DNA replication at telomeres. In this study, we have demonstrated that ORC subunits can be recruited to telomere-repeat DNA in nuclear extracts by DNA affinity purification and in vivo by ChIP assay. We also demonstrate that ORC2 can colocalize with a subset of TRF2 and to telomere-repeat foci in a TRF2-dependent manner and that this association could be disrupted by the overexpression of TRF2DB. We showed that ORC2-depleted cells have an accelerated loss of telomere-repeat signals and form telomere circles similar to cells overexpressing TRF2DB. These data suggest that ORC is at least one functional target of the TRF2 basic amino-terminal domain. Because ORC was found at w40%–60% of telomere foci, it is also possible that ORC interaction with TRF2 and its localization at telomeres are subject to cell-cycle or telomere-structure-dependent regulation.
(G) Quantification of observed cytological alterations observed in siRNA-transfected HCT116 cells or in untransfected e83 and control parental cell lines. Numbers above bars represent total telomere-signal-free ends or TDMs out of the total number of counted chromosome ends or chromosomes, respectively.
Current Biology Vol 17 No 22 1994
Figure 4. ORC2 Depletion Increases Telomere-Circle Formation (A–E) Telomere-repeat restriction digest (AluI/MboI) fragments were analyzed by Southern blotting of 2D neutral agarose gels. Telomere circles are indicated by arrows, and slow-mobility DNA is indicated by arrowheads. (A) HCT116 cells were transfected three times for 7 days with siControl, siORC2-1, or siORC2-2, analyzed by Southern blotting of 2D gels, and detected with TTAGGG (top panels) or Alu (lower panels)-specific probes. Molecular-weight markers are indicated for the first dimension. A quantification of telomere circles relative to linear DNA is indicated below. (B) ORC2D/2 and parental wild-type controls were analyzed for telomere-circle formation as described in (A). (C) HCT116 cells were untreated (2) or treated with 50 mM HU for 4 days and then analyzed by 2D neutral agarose gels. (D) U2OS cells transfected with siControl (left) or siORC-1 (right) were analyzed by 2D gel for telomere-repeat DNA (TTAGGG) (top panel) or with Alu probe as an internal loading control (Alu). (E) Same as in (C), except in U2OS cells. Ligated lambda HinDIII DNA fragments were used for markers of linear and circular forms of DNA (l DNA). The merge combines the images of the telomere and l DNA hybridization.
What then is the function of ORC at telomeres? ORC is multifunctional and has established roles in DNA-replication initiation, transcription silencing, heterochromatin formation, and sister-chromatid cohesion [3, 4]. All of these ORC functions might be important at telomeres. It remains unclear whether ORC nucleates active origins of bidirectional DNA replication within telomere repeats or in adjacent subtelomeric sequences, which might
also have TRF2-binding sites. ORC-binding sites and active origins of DNA replication are known to exist in the subtelomeres of yeast [9, 26] but have not been demonstrated in higher eukaryotes or in the TTAGGG tracts at telomere termini. A recent study found that the TRF2 ortholog, TAZ1, is required for the complete semiconservative DNA replication of telomeres [27]. We propose that TRF2, like TAZ1, might facilitate DNA replication by
ORC Functions in Telomere Maintenance 1995
recruitment of ORC subunits to telomere-repeat tracts in subtelomeres or in long TTAGGG tracts that potentially challenge replication machinery. We propose that ORC recruitment is required for the formation of replication origins and the completion of semiconservative DNA replication at telomeres. However, it is also possible that ORC facilitates heterochromatin formation or sister-chromatid cohesion, which might also be necessary for telomere-repeat stability. Because telomere T loops share some structural similarity to replication origins, it is also possible that ORC performs a common function in the formation and maintenance of these related DNA structures. Supplemental Data Experimental Procedures and ten figures are available at http:// www.current-biology.com/cgi/content/full/17/22/1989/DC1/. Acknowledgments We thank Harold Riethman and Titia de Lange for plasmids, Jan Karlseder for telomere FISH protocols, Akira Nabetani for 2D electrophoresis protocols, and Illja Demuth for Rabbit polyclonal SNM1b antibody. We also acknowledge the contribution of the Wistar Institute Cancer Center Flow Cytometry (Jeffery Faust), Microscopy (James Hayden), and Proteomics Core Facilities (David Speicher and Tom Beer). This work was supported by a Leukemia & Lymphoma Society of America Special Fellow award (Z. D.) and by the National Institutes of Health (CA093606). Received: May 30, 2007 Revised: October 5, 2007 Accepted: October 9, 2007 Published online: November 15, 2007 References 1. de Lange, T. (2005). Shelterin: The protein complex that shapes and safeguards human telomeres. Genes Dev. 19, 2100–2110. 2. Munoz, P., Blanco, R., and Blasco, M.A. (2006). Role of the TRF2 telomeric protein in cancer and ageing. Cell Cycle 5, 718–721. 3. Chesnokov, I.N. (2007). Multiple functions of the origin recognition complex. Int. Rev. Cytol. 256, 69–109. 4. Bell, S.P. (2002). The origin recognition complex: From simple origins to complex functions. Genes Dev. 16, 659–672. 5. Vega, L.R., Mateyak, M.K., and Zakian, V.A. (2003). Getting to the end: Telomerase access in yeast and humans. Nat. Rev. Mol. Cell Biol. 4, 948–959. 6. Blackburn, E.H., Greider, C.W., and Szostak, J.W. (2006). Telomeres and telomerase: The path from maize, Tetrahymena and yeast to human cancer and aging. Nat. Med. 12, 1133–1138. 7. de Lange, T. (2002). Protection of mammalian telomeres. Oncogene 21, 532–540. 8. de Lange, T. (2004). T-loops and the origin of telomeres. Nat. Rev. Mol. Cell Biol. 5, 323–329. 9. McEachern, M.J., and Haber, J.E. (2006). Break-induced replication and recombinational telomere elongation in yeast. Annu. Rev. Biochem. 75, 111–135. 10. Karlseder, J. (2006). Telomeric proteins: Clearing the way for the replication fork. Nat. Struct. Mol. Biol. 13, 386–387. 11. Smogorzewska, A., and de Lange, T. (2004). Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. 73, 177–208. 12. Bell, S.P., and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374. 13. Prasanth, S.G., Mendez, J., Prasanth, K.V., and Stillman, B. (2004). Dynamics of pre-replication complex proteins during the cell division cycle. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 7–16. 14. Makovets, S., Herskowitz, I., and Blackburn, E.H. (2004). Anatomy and dynamics of DNA replication fork movement in yeast telomeric regions. Mol. Cell. Biol. 24, 4019–4031.
15. Atanasiu, C., Deng, Z., Wiedmer, A., Norseen, J., and Lieberman, P.M. (2006). ORC binding to TRF2 stimulates OriP replication. EMBO Rep. 7, 716–721. 16. Shareef, M.M., Badugu, R., and Kellum, R. (2003). HP1/ORC complex and heterochromatin assembly. Genetica 117, 127– 134. 17. Prasanth, S.G., Prasanth, K.V., Siddiqui, K., Spector, D.L., and Stillman, B. (2004). Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. EMBO J. 23, 2651–2663. 18. Pak, D.T., Pflumm, M., Chesnokov, I., Huang, D.W., Kellum, R., Marr, J., Romanowski, P., and Botchan, M.R. (1997). Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91, 311–323. 19. van Overbeek, M., and de Lange, T. (2006). Apollo, an Artemisrelated nuclease, interacts with TRF2 and protects human telomeres in S phase. Curr. Biol. 16, 1295–1302. 20. Lenain, C., Bauwens, S., Amiard, S., Brunori, M., Giraud-Panis, M.J., and Gilson, E. (2006). The Apollo 50 exonuclease functions together with TRF2 to protect telomeres from DNA repair. Curr. Biol. 16, 1303–1310. 21. Smogorzewska, A., van Steensel, B., Bianchi, A., Oelmann, S., Schaefer, M.R., Schnapp, G., and de Lange, T. (2000). Control of human telomere length by TRF1 and TRF2. Mol. Cell. Biol. 20, 1659–1668. 22. Wang, R.C., Smogorzewska, A., and de Lange, T. (2004). Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119, 355–368. 23. Dhar, S.K., Yoshida, K., Machida, Y., Khaira, P., Chaudhuri, B., Wohlschlegel, J.A., Leffak, M., Yates, J., and Dutta, A. (2001). Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin. Cell 106, 287–296. 24. Zhu, X.D., Niedernhofer, L., Kuster, B., Mann, M., Hoeijmakers, J.H., and de Lange, T. (2003). ERCC1/XPF removes the 30 overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes. Mol. Cell 12, 1489–1498. 25. Shimada, K., and Gasser, S.M. (2007). The origin recognition complex functions in sister-chromatid cohesion in Saccharomyces cerevisiae. Cell 128, 85–99. 26. Loo, S., and Rine, J. (1995). Silencing and heritable domains of gene expression. Annu. Rev. Cell Dev. Biol. 11, 519–548. 27. Miller, K.M., Rog, O., and Cooper, J.P. (2006). Semi-conservative DNA replication through telomeres requires Taz1. Nature 440, 824–828.
DOI 10.1016/j.cub.2007.10.054
Report The Origin Recognition Complex Localizes to Telomere Repeats and Prevents Telomere-Circle Formation Zhong Deng,1 Jayaraju Dheekollu,1 Dominique Broccoli,2 Anindya Dutta,3 and Paul M. Lieberman1,* 1The Wistar Institute Philadelphia, Pennsylvania 19104 2Memorial Health University Medical Center Savannah, Georgia 31404 3University of Virginia Charlottesville, Virginia 22908
Summary Chromosome ends are maintained by telomere-repeat-binding factors (TRFs) that coordinate DNA end protection with telomere replication [1, 2]. The origin recognition complex (ORC) coordinates bidirectional DNA replication at most chromosomal sites, but it is also known to function in transcriptional silencing, heterochromatin formation, and sister-chromatid cohesion [3, 4]. We now show that ORC localizes to telomere repeats and contributes to telomere maintenance. We found that ORC subunits can be affinity purified with telomere-repeat DNA along with other components of the known ‘‘shelterin’’ complex. ORC subunits colocalized with telomere-repeat foci and coimmunoprecipitated with TRF2 but not TRF2 lacking its amino-terminal basic domain (TRF2DB). ORC2 depletion or hypomorphic cell lines caused a loss of telomere-repeat signal intensity and the appearance of dysfunctional telomeres, including telomere-signalfree ends and telomere-repeat-containing double minutes. Two-dimensional agarose gel electrophoresis revealed that ORC2 depletion increased telomere circle formation, comparable to the overexpression of TRF2DB. A similar increase in telomere circle formation was induced by hydroxyurea treatment, providing evidence that replication stress produces telomere circles. These findings suggest that ORC recruitment by TRF2 contributes to telomere integrity by facilitating efficient telomere DNA replication and preventing the generation of telomere-repeat-containing circles. Results and Discussion The completion of DNA replication at the ends of linear chromosomes can be problematic not only because of the end-replication problem but also as a result of repetitive DNA and nucleoprotein-structures that might act as replication fork barriers [5, 6]. The telomere repeats and the proteins that associate with these repeats provide chromosome ends with protection from nucleolytic degradation and end-to-end fusions that would otherwise lead to genetic instability [7, 8]. Telomere-repeat length is maintained by the interplay of telomerase,
*Correspondence: [email protected]
recombination-based lengthening, and semiconservative DNA replication [9, 10]. The telomere-repeat-binding factors that protect telomeres from degradation and fusions are also important for maintaining telomere length and coordinating the access of replication proteins to telomere DNA [10, 11]. It is generally thought that most replication initiates outside of telomere repeats at chromosomal origins bound by ORC [12–14]. In lower eukaryotes, ORC is known to bind to specific sites in the subtelomere repeats. However, in higher eukaryotes ORC lacks sequence-specific DNA binding, and it is not clear how it is recruited to specific chromosomal regions. In an earlier study, we found that TRF2 facilitated ORC recruitment to a viral origin of DNA replication [15]. This earlier study raised the question of whether TRF2 recruited ORC to cellular telomeres and whether ORC plays a role in telomere maintenance. To investigate the possibility that ORC bound to telomere-repeat DNA, we isolated nuclear factors that bound to telomere repeats by using DNA affinity chromatography. Proteins from Raji cell nuclear extracts were purified with biotinylated DNA containing 12 telomere repeats (TTAGGG) or control (BKS) DNA bound to streptavidin magnetic beads. Affinity-purified proteins were visualized by colloidal blue staining and then identified by mass spectrometry (Figures 1A and 1B). The major proteins isolated were among those already known to bind directly to telomere repeats, including TRF2, hRap1, and TRF1 [1]. Other members of the telomere-associated shelterin complex, including TIN2, POT1, and TPP1, were also identified [1]. Notably, we identified four of the six ORC subunits in the telomere-repeat DNA affinity-purified samples. ORC1, ORC2, and ORC4 binding specificity for telomere repeats was verified by western blotting (Figure 1B). Heterochromatin protein 1 (HP1a) [16], which has been reported to interact with ORC [17, 18], was weakly detected by western blot, as was the recently identified telomere-associated 50 exonuclease SNM1B (also referred to as Apollo) [19, 20]. ORC2 is an essential core subunit of ORC and was further analyzed for its association with telomere repeats in vivo by chromatin immunoprecipitation (ChIP) (Figure 1C). Telomere-repeat DNA was enriched in ORC2-specific ChIP (3.6% input) but not in ChIP with control IgG (<0.4% input) or antibodies to the EBV-encoded EBNA1 protein that binds OriP. ORC2 bound telomere-repeat DNA to a greater extent than to a well-characterized viral origin of DNA replication (DS) or to cellular GAPDH DNA. The interaction of ORC2 with telomere DNA was also observed to varying extents in cells with different telomere-maintenance mechanisms, including telomerase-positive HCT and HeLa, telomerase-negative cells U2OS, which maintain long telomeres by a recombinational mechanism referred to as alternative lengthening of telomeres (ALT), and telomerase-negative primary human peripheral blood mononuclear cells (PBMCs). These findings indicate that ORC2 associates with telomere-repeat DNA in multiple cell types,
Current Biology Vol 17 No 22 1990
Figure 1. ORC Associates with Telomere-Repeat DNA (A) DNA affinity purification of Raji nuclear extract with (TTAGGG)12 or control BKS DNA. Purified proteins were visualized with colloidal blue and identified with LC/MS/MS. (B) We used western-blot analysis to confirm the identity of proteins identified by LC/MS/MS. Proteins boxed in green were considered negative controls. ORC subunits are boxed in red. (C) ChIP assays with antibodies specific to TRF2, EBNA1, and ORC2 were probed with telomere-repeat DNA (TTAGGG, left panel), EBV OriP DNA (DS, middle panel), or control GAPDH DNA (right panel) in Raji cells. A quantification of three independent ChIP dot blots is shown below. (D) ChIP analysis of ORC2 or IgG control antibodies at TTAGGG (top) or Alu (bottom) sequence in Raji, HCT, HeLA, U2OS, or peripheral blood mononuclear cells from healthy donors (PBMC). Error bars represent mean values (6SD). (E) Indirect immunofluorescence (IF) was performed with antibody to ORC2 (red) and TRF2 (green) in HCT116 (Ei–Eiv) or U2OS (Ei0 –Eiv0 ) cells. Merge of ORC2 and TRF2 was combined with DAPI (Eiii and Eiii0 ) and enlarged (Eiv and Eiv0 ). The average percentage of TRF2 foci that colocalize with ORC2 foci is indicated to the right, where n is the number of images counted. (F) Immuno-FISH was performed with antibody to ORC2 (red) and TTAGGG PNA probe (green) in HCT116 or U2OS.
independent of the telomere-lengthening mechanism (Figure 1D, top panel). The association was specific for telomere-repeat DNA because ORC2 ChIP was not enriched for Alu-repeat DNA (Figure 1D, bottom panel). A
similar enrichment of ORC at telomere repeats was observed when FLAG-ORC2 or FLAG-ORC1 was ectopically expressed in HCT116 cells (Figure S1C in the Supplemental Data available online), indicating that multiple
ORC Functions in Telomere Maintenance 1991
Figure 2. TRF2 Is Required for ORC Recruitment at Telomeres (A) siTRF2 or control siRNA was transfected into HCT116 cells and assayed at 72 hr after transfection by western blotting for TRF2, ORC2, or PCNA expression levels. (B) siControl or siTRF2-transfected HCT116 cells were analyzed by ChIP assay in duplicates with anti-TRF2, anti-ORC2, or IgG control and were probed by hybridization with TTAGGG (left panel) or Alu repeats (right panel). (C) Quantification of four independent experiments represented in (B). (D) FLAG-TRF2 (top panels) or FLAG-TRF2DB (lower panels) was transfected into HCT116 cells, immunoprecipitated, and assayed by western blotting with antibodies to FLAG (top) or ORC2 (bottom). (E) TRF2DB transfected HCT116 cells or control cells were assayed by ChIP 72 hr after transfection with antibodies to TRF2, ORC2, or control IgG and probed with telomere TTAGGG, Alu repeat, or single-copy gene 36BD4 DNA. (F) Quantification of three independent ChIP assays is shown in (E).
ORC subunits associate with telomere repeats. Also, siRNA depletion of ORC2 reduced ORC2 ChIP at telomeres, further indicating that ORC2 antibodies were specific for ORC2 at telomeres (Figure S2). We next tested whether ORC2 protein colocalized with TRF2-associated telomere foci by using indirect immunofluorescence (IF) (Figures 1E and 1F). After detergent extraction that enriches for telomere-specific foci, we found that w50% of the TRF2 foci in HCT116 and w65% of the TRF2 foci in U2OS cells colocalized with ORC2 foci (Figure 1E). To determine whether the ORC2 foci colocalized with telomere-repeat DNA, we performed immuno-FISH with anti-ORC2 antibody and a TTAGGG PNA probe (Figure 1F). We observed nearly identical results to those found with TRF2 foci, in which ORC2 colocalized with w44% or w66% of the TTAGGG foci in HCT116 or U2OS cells, respectively. This colocalization was not limited to ORC2; transfected GFP-ORC1 also colocalized with TRF2-specific telomere foci to a similar extent as ORC2, in both HCT and U2OS cells (Figure S3). Additionally, DsRed-TRF2 colocalized with w68% of the GFP-ORC1 foci in transfected cells, indicating that the colocalization was not a consequence of antibody crossreactivity (Figure S4). These findings indicate that ORC1 and ORC2 associate with approximately half
of the telomere-repeat foci in telomerase-positive and ALT-positive cell lines. Previous studies demonstrated that TRF2 can interact with ORC subunits [15] (also Figures S1A and S1B). To determine whether TRF2 was required for ORC recruitment to telomeres, we tested whether ORC associated with telomere-repeat DNA in cells in which TRF2 was depleted by siRNA (Figures 2A–2C). TRF2 was efficiently depleted in HCT116 cells transfected with siTRF2 but not in cells transfected with control siRNA (Figure 2A). siTRF2 had no detectable effect on ORC2 or PCNA protein levels in transient transfection assays 72 hr after transfection. By using ChIP assays, we found that TRF2 depletion reduced ORC2 interaction with telomererepeat DNA by w5-fold (Figures 2B and 2C). A similar result was observed by IF, during which siRNA depletion of TRF2 abrogated ORC2 colocalization with TRF1 telomere foci (Figure S5). Previous studies demonstrated that TRF2 amino-terminal domain was important for interactions with ORC [15]. These findings were further substantiated by coimmunoprecipitation experiments that show ORC2 interacts with full-length TRF2 but not with TRF2 lacking the amino-terminal basic domain (TRF2DB) (Figure 2D). We next asked whether TRF2DB overexpression disrupts ORC association
Current Biology Vol 17 No 22 1992
Figure 3. Telomere-Repeat Loss and Dysfunction in ORC2 Depleted Cells (A) HCT116 cells were harvested at day 7 after three separate transfections with control, ORC2-1, or ORC2-2 siRNAs. Telomere-repeat length and intensity was measured by restriction digest of genomic DNA with AluI/MboI and Southern hybridization with DIG-labeled (TTAGGG)6 probe (middle panel) or control Alu (right panel). Ethidium-bromide staining of total DNA digest was used as a control for DNA loading (left panel). The position of MWs (kb) is indicated to the left. (B) Western blot of ORC2 in siControl, siORC2-1-, and siORC2-2-treated HCT116 cells used in the experiments shown in Figure 3A. TRF2, PCNA, and p54nrb blots were used as loading controls. (C) Relative telomere-repeat-signal loss was quantified for at least four independent experiments (Figure S7 and data not shown). (D) Telomere-repeat length of parental HCT116 and the ORC2 hypomorphic cell line e83 was measured as above at indicated time points (left panel). The right panel shows the telomere-repeat length of HCT116 cells transfected with siControl or siORC2-specific siRNA at various days after transfection. Arrowheads indicate approximate median telomere-repeat lengths of wild-type or control siRNAtransfected cells, and brackets represent median telomere signals of ORC2 hypomorphic or depleted cells. (E) Representative telomere FISH analysis on metaphase spreads for telomere defects. siRNA-transfected cells were harvested on day 7 posttransfection. White arrows indicate common telomere aberrations. (F) Magnified view of common telomere aberrations found in e83 or ORC2 depleted cells: telomere-signal-free ends (Fi–Fiii), telomere-containing double minutes (TDM, Fiv–Fvii), and sister chromatid lacking cohesion (Fviii and Fix).
ORC Functions in Telomere Maintenance 1993
with telomere-repeat DNA (Figures 2E and 2F). TRF2DB overexpression caused a w2.5-fold decrease in ORC association with telomere-repeat DNA by ChIP assay (Figure 2E, left panel). We also found that FLAG-TRF2DB failed to colocalize with ORC2 foci, whereas FLAG-TRF2 wild-type colocalized with ORC2 in IF experiments (Figure S6). These findings support the model that the TRF2 amino-terminal domain is required for ORC recruitment to telomeres [15]. The functional consequences of TRFDB overexpression and TRF2 depletion on telomere maintenance have been characterized previously [21, 22]. In HCT116 cells, we found that TRF2DB overexpression led to a loss of telomere-repeat signal intensity by w24% in restriction fragment-length assays (Figure S7E). We reasoned that if ORC was a functionally relevant target of TRF2 amino-terminal basic domain, then ORC2 depletion should have a similar effect as overexpression of TRF2DB. Two different siRNA, siORC2-1 and siORC2-2, efficiently depleted ORC2 but not control proteins TRF2, PCNA, or p54nrb from HCT116 cells (Figure 3B and Figure S8B). siORC2-1 had no significant effect on cell-cycle profile and cell proliferation, whereas siORC22 produced a slight increase in sub-G1 cells, but cells continued to proliferate at comparable rates (Figure S8C and data not shown). Repetitive transfection with siORC2-1 and siORC2-2 caused a w21 and 27% loss, respectively, of telomere-repeat signal intensity relative to control siRNA (Figure 3A). Similar loss of telomere-repeat signals by siORC2-1 was also observed in U2OS cells (Figure S7F). To eliminate the possibility that siORC depletion was due to off-target effects, we analyzed the telomere-repeat signals in an HCT116-derived cell line, referred to as e83, that is hypomorphic for ORC2 (ORC2D/2) [23]. We found that telomere-repeat signal in e83 was weaker and was lost at an accelerated rate relative to parental HCT116 cells (Figure 3D, left panel, and Figure S8D for loading controls). This accelerated loss of telomere-repeat signal was also observed when HCT116 cells were repeatedly transfected with siORC2-1, but not with control siRNA (Figure 3B, right panel and Figure S8E for loading controls; see also Figures S7B and S7C). These data indicate that ORC2 depletion by siRNA or hypomorphic cell lines leads to loss of telomere repeat DNA. This loss of telomere repeat signal is comparable to that observed when TRF2DB is overexpressed. To better assess the effect of ORC2 on individual telomere signals, we examined metaphase chromosome telomeres by fluorescence in situ hybridization (FISH) in siRNA-depleted and hypomorphic ORC2 cells (Figures 3E–3G). We found that reduced ORC2 levels resulted in several cytological telomere aberrations, including a 4- to 6-fold increase in signal-free ends and a 6- to 12fold increase in telomere-containing double minutes (Figure 3G). Overexpression of TRF2DB is also known to increase signal-free ends [22], suggesting that ORC2 might cooperate with TRF2 in maintaining telomere repeats. Double minutes containing telomere repeats were previously found enriched in cells depleted in
ERCC1 [24], but it is not clear whether ORC2 interacts with components of the ERCC1 pathway at telomeres. Interestingly, we also observed an increase in separated sister chromatids (Figures 3Fviii and 3Fix), suggesting that mammalian ORC might function in sister-chromatid cohesion similar to that described for budding yeast [25]. Overexpression of TRF2DB is known to cause telomere-circle formation [22]. The loss of a telomere-repeat signals and the accumulation of telomere cytological defects (Figure 3) suggests that ORC depletion might also cause telomere-circle formation. To test this possibility, we analyzed telomere-repeat DNA by 2D neutral agarose gel electrophoresis and Southern-blot analysis (Figure 4). We found that siORC2-1 and siORC2-2 increased the formation of telomere-repeat-circle arcs relative to linear arcs by w6.5-fold in HCT116 cells (Figure 4A). The telomere-circle arcs comigrated with self-ligated lambda DNA (Figures S9 and S10) and were detected equally with TTAGGG and CCCTAA probes (data not shown), indicating that the arcs consist of circular, doublestranded telomere DNA (data not shown). No circle arcs were observed at Alu-repeat DNA (Figure 4A, lower panel) indicating that ORC2 depletion increased telomere-circle formation specifically (Figures S9 and S10). Telomere circles were also observed at higher frequency (w2.9 fold) in ORC2 hypomorphic cell line e83 relative to HCT parental controls (Figure 4B) and in ORC2-depleted U2OS cells (w1.5-fold), which are known to have significant background levels of telomere circles (Figure 4D). We also noted that a slower migrating form of telomeric DNA accumulated in ORC2 depleted and hypomorphic cells (indicated by the arrowheads). These slow-migrating molecules might correspond to complex DNA structures, including replication and recombination intermediates. To determine whether ORC2 depletion induced telomere circles as a consequence of incomplete replication, we assayed telomere DNA structure in cells treated with low doses of the ribonucleotide reductase inhibitor hydroxyurea (HU) (Figures 4C and 4E). We found that HU treatment induced telomere-circle formation and the appearance of slow-mobility structures, similar to ORC2 depletion. This suggests that telomere-circle formation can be caused by incomplete DNA replication at telomeres. In this study, we have demonstrated that ORC subunits can be recruited to telomere-repeat DNA in nuclear extracts by DNA affinity purification and in vivo by ChIP assay. We also demonstrate that ORC2 can colocalize with a subset of TRF2 and to telomere-repeat foci in a TRF2-dependent manner and that this association could be disrupted by the overexpression of TRF2DB. We showed that ORC2-depleted cells have an accelerated loss of telomere-repeat signals and form telomere circles similar to cells overexpressing TRF2DB. These data suggest that ORC is at least one functional target of the TRF2 basic amino-terminal domain. Because ORC was found at w40%–60% of telomere foci, it is also possible that ORC interaction with TRF2 and its localization at telomeres are subject to cell-cycle or telomere-structure-dependent regulation.
(G) Quantification of observed cytological alterations observed in siRNA-transfected HCT116 cells or in untransfected e83 and control parental cell lines. Numbers above bars represent total telomere-signal-free ends or TDMs out of the total number of counted chromosome ends or chromosomes, respectively.
Current Biology Vol 17 No 22 1994
Figure 4. ORC2 Depletion Increases Telomere-Circle Formation (A–E) Telomere-repeat restriction digest (AluI/MboI) fragments were analyzed by Southern blotting of 2D neutral agarose gels. Telomere circles are indicated by arrows, and slow-mobility DNA is indicated by arrowheads. (A) HCT116 cells were transfected three times for 7 days with siControl, siORC2-1, or siORC2-2, analyzed by Southern blotting of 2D gels, and detected with TTAGGG (top panels) or Alu (lower panels)-specific probes. Molecular-weight markers are indicated for the first dimension. A quantification of telomere circles relative to linear DNA is indicated below. (B) ORC2D/2 and parental wild-type controls were analyzed for telomere-circle formation as described in (A). (C) HCT116 cells were untreated (2) or treated with 50 mM HU for 4 days and then analyzed by 2D neutral agarose gels. (D) U2OS cells transfected with siControl (left) or siORC-1 (right) were analyzed by 2D gel for telomere-repeat DNA (TTAGGG) (top panel) or with Alu probe as an internal loading control (Alu). (E) Same as in (C), except in U2OS cells. Ligated lambda HinDIII DNA fragments were used for markers of linear and circular forms of DNA (l DNA). The merge combines the images of the telomere and l DNA hybridization.
What then is the function of ORC at telomeres? ORC is multifunctional and has established roles in DNA-replication initiation, transcription silencing, heterochromatin formation, and sister-chromatid cohesion [3, 4]. All of these ORC functions might be important at telomeres. It remains unclear whether ORC nucleates active origins of bidirectional DNA replication within telomere repeats or in adjacent subtelomeric sequences, which might
also have TRF2-binding sites. ORC-binding sites and active origins of DNA replication are known to exist in the subtelomeres of yeast [9, 26] but have not been demonstrated in higher eukaryotes or in the TTAGGG tracts at telomere termini. A recent study found that the TRF2 ortholog, TAZ1, is required for the complete semiconservative DNA replication of telomeres [27]. We propose that TRF2, like TAZ1, might facilitate DNA replication by
ORC Functions in Telomere Maintenance 1995
recruitment of ORC subunits to telomere-repeat tracts in subtelomeres or in long TTAGGG tracts that potentially challenge replication machinery. We propose that ORC recruitment is required for the formation of replication origins and the completion of semiconservative DNA replication at telomeres. However, it is also possible that ORC facilitates heterochromatin formation or sister-chromatid cohesion, which might also be necessary for telomere-repeat stability. Because telomere T loops share some structural similarity to replication origins, it is also possible that ORC performs a common function in the formation and maintenance of these related DNA structures. Supplemental Data Experimental Procedures and ten figures are available at http:// www.current-biology.com/cgi/content/full/17/22/1989/DC1/. Acknowledgments We thank Harold Riethman and Titia de Lange for plasmids, Jan Karlseder for telomere FISH protocols, Akira Nabetani for 2D electrophoresis protocols, and Illja Demuth for Rabbit polyclonal SNM1b antibody. We also acknowledge the contribution of the Wistar Institute Cancer Center Flow Cytometry (Jeffery Faust), Microscopy (James Hayden), and Proteomics Core Facilities (David Speicher and Tom Beer). This work was supported by a Leukemia & Lymphoma Society of America Special Fellow award (Z. D.) and by the National Institutes of Health (CA093606). Received: May 30, 2007 Revised: October 5, 2007 Accepted: October 9, 2007 Published online: November 15, 2007 References 1. de Lange, T. (2005). Shelterin: The protein complex that shapes and safeguards human telomeres. Genes Dev. 19, 2100–2110. 2. Munoz, P., Blanco, R., and Blasco, M.A. (2006). Role of the TRF2 telomeric protein in cancer and ageing. Cell Cycle 5, 718–721. 3. Chesnokov, I.N. (2007). Multiple functions of the origin recognition complex. Int. Rev. Cytol. 256, 69–109. 4. Bell, S.P. (2002). The origin recognition complex: From simple origins to complex functions. Genes Dev. 16, 659–672. 5. Vega, L.R., Mateyak, M.K., and Zakian, V.A. (2003). Getting to the end: Telomerase access in yeast and humans. Nat. Rev. Mol. Cell Biol. 4, 948–959. 6. Blackburn, E.H., Greider, C.W., and Szostak, J.W. (2006). Telomeres and telomerase: The path from maize, Tetrahymena and yeast to human cancer and aging. Nat. Med. 12, 1133–1138. 7. de Lange, T. (2002). Protection of mammalian telomeres. Oncogene 21, 532–540. 8. de Lange, T. (2004). T-loops and the origin of telomeres. Nat. Rev. Mol. Cell Biol. 5, 323–329. 9. McEachern, M.J., and Haber, J.E. (2006). Break-induced replication and recombinational telomere elongation in yeast. Annu. Rev. Biochem. 75, 111–135. 10. Karlseder, J. (2006). Telomeric proteins: Clearing the way for the replication fork. Nat. Struct. Mol. Biol. 13, 386–387. 11. Smogorzewska, A., and de Lange, T. (2004). Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. 73, 177–208. 12. Bell, S.P., and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374. 13. Prasanth, S.G., Mendez, J., Prasanth, K.V., and Stillman, B. (2004). Dynamics of pre-replication complex proteins during the cell division cycle. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 7–16. 14. Makovets, S., Herskowitz, I., and Blackburn, E.H. (2004). Anatomy and dynamics of DNA replication fork movement in yeast telomeric regions. Mol. Cell. Biol. 24, 4019–4031.
15. Atanasiu, C., Deng, Z., Wiedmer, A., Norseen, J., and Lieberman, P.M. (2006). ORC binding to TRF2 stimulates OriP replication. EMBO Rep. 7, 716–721. 16. Shareef, M.M., Badugu, R., and Kellum, R. (2003). HP1/ORC complex and heterochromatin assembly. Genetica 117, 127– 134. 17. Prasanth, S.G., Prasanth, K.V., Siddiqui, K., Spector, D.L., and Stillman, B. (2004). Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. EMBO J. 23, 2651–2663. 18. Pak, D.T., Pflumm, M., Chesnokov, I., Huang, D.W., Kellum, R., Marr, J., Romanowski, P., and Botchan, M.R. (1997). Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91, 311–323. 19. van Overbeek, M., and de Lange, T. (2006). Apollo, an Artemisrelated nuclease, interacts with TRF2 and protects human telomeres in S phase. Curr. Biol. 16, 1295–1302. 20. Lenain, C., Bauwens, S., Amiard, S., Brunori, M., Giraud-Panis, M.J., and Gilson, E. (2006). The Apollo 50 exonuclease functions together with TRF2 to protect telomeres from DNA repair. Curr. Biol. 16, 1303–1310. 21. Smogorzewska, A., van Steensel, B., Bianchi, A., Oelmann, S., Schaefer, M.R., Schnapp, G., and de Lange, T. (2000). Control of human telomere length by TRF1 and TRF2. Mol. Cell. Biol. 20, 1659–1668. 22. Wang, R.C., Smogorzewska, A., and de Lange, T. (2004). Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119, 355–368. 23. Dhar, S.K., Yoshida, K., Machida, Y., Khaira, P., Chaudhuri, B., Wohlschlegel, J.A., Leffak, M., Yates, J., and Dutta, A. (2001). Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin. Cell 106, 287–296. 24. Zhu, X.D., Niedernhofer, L., Kuster, B., Mann, M., Hoeijmakers, J.H., and de Lange, T. (2003). ERCC1/XPF removes the 30 overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes. Mol. Cell 12, 1489–1498. 25. Shimada, K., and Gasser, S.M. (2007). The origin recognition complex functions in sister-chromatid cohesion in Saccharomyces cerevisiae. Cell 128, 85–99. 26. Loo, S., and Rine, J. (1995). Silencing and heritable domains of gene expression. Annu. Rev. Cell Dev. Biol. 11, 519–548. 27. Miller, K.M., Rog, O., and Cooper, J.P. (2006). Semi-conservative DNA replication through telomeres requires Taz1. Nature 440, 824–828.