Abstract
Telomere maintenance is critical for genome stability. The newly-identified Ctc1/Stn1/Ten1 complex is important for telomere maintenance, though its precise role is unclear. We report here that depletion of hStn1 induces catastrophic telomere shortening, DNA damage response, and early senescence in human somatic cells. These phenotypes are likely due to the essential role of hStn1 in promoting efficient replication of lagging-strand telomeric DNA. Downregulation of hStn1 accumulates single-stranded G-rich DNA specifically at lagging-strand telomeres, increases telomere fragility, hinders telomere DNA synthesis, as well as delays and compromises telomeric C-strand synthesis. We further show that hStn1 deficiency leads to persistent and elevated association of DNA polymerase α (polα) to telomeres, suggesting that hStn1 may modulate the DNA synthesis activity of polα rather than controlling the loading of polα to telomeres. Additionally, our data suggest that hStn1 is unlikely to be part of the telomere capping complex. We propose that the hStn1 assists DNA polymerases to efficiently duplicate lagging-strand telomeres in order to achieve complete synthesis of telomeric DNA, therefore preventing rapid telomere loss.
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References
Palm W, de Lange T . How shelterin protects mammalian telomeres. Annu Rev Genet 2008; 42:301–334.
Miyake Y, Nakamura M, Nabetani A, et al. RPA-like mammalian Ctc1-Stn1-Ten1 complex binds to single-stranded DNA and protects telomeres independently of the Pot1 pathway. Mol Cell 2009; 36:193–206.
Surovtseva YV, Churikov D, Boltz KA, et al. Conserved telomere maintenance component 1 interacts with STN1 and maintains chromosome ends in higher eukaryotes. Mol Cell 2009; 36:207–218.
Dai X, Huang C, Bhusari A, Sampathi S, Schubert K, Chai W . Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection. EMBO J 2010; 29:2788–2801.
Wan M, Qin J, Songyang Z, Liu D . OB-fold containing protein 1 (OBFC1), a human homologue of yeast Stn1, associates with TPP1 and is implicated in telomere length regulation. J Biol Chem 2009; 284:26725–26731.
Casteel DE, Zhuang S, Zeng Y, et al. A DNA polymerase-{alpha}{middle dot}primase cofactor with homology to replication protein A-32 regulates DNA replication in mammalian cells. J Biol Chem 2009; 284:5807–5818.
Levy D, Neuhausen SL, Hunt SC, et al. Genome-wide association identifies OBFC1 as a locus involved in human leukocyte telomere biology. Proc Natl Acad Sci USA 2010; 107:9293–9298.
Anderson BH, Kasher PR, Mayer J, et al. Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus. Nat Genet 2012; 44:338–342.
Polvi A, Linnankivi T, Kivela T, et al. Mutations in CTC1, encoding the CTS telomere maintenance complex component 1, cause cerebroretinal microangiopathy with calcifications and cysts. Am J Hum Genet 2012; 90:540–549.
Keller RB, Gagne KE, Usmani GN, et al. CTC1 Mutations in a patient with dyskeratosis congenita. Pediatr Blood Cancer 2012; 59:311–314.
Martin V, Du LL, Rozenzhak S, Russell P . Protection of telomeres by a conserved Stn1-Ten1 complex. Proc Natl Acad Sci USA 2007; 104:14038–14043.
Song X, Leehy K, Warrington RT, Lamb JC, Surovtseva YV, Shippen DE . STN1 protects chromosome ends in Arabidopsis thaliana. Proc Natl Acad Sci USA 2008; 105:19815–19820.
Grandin N, Damon C, Charbonneau M . Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination. EMBO J 2001; 20:6127–6139.
Grandin N, Damon C, Charbonneau M . Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13. EMBO J 2001; 20:1173–1183.
Qi H, Zakian VA . The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase alpha and the telomerase-associated est1 protein. Genes Dev 2000; 14:1777–1788.
Chandra A, Hughes TR, Nugent CI, Lundblad V . Cdc13 both positively and negatively regulates telomere replication. Genes Dev 2001; 15:404–414.
Grandin N, Damon C, Charbonneau M . Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment. Mol Cell Biol 2000; 20:8397–8408.
Grandin N, Reed SI, Charbonneau M . Stn1, a new Saccharomyces cerevisiae protein, is implicated in telomere size regulation in association with Cdc13. Genes Dev 1997; 11:512–527.
Nugent CI, Hughes TR, Lue NF, Lundblad V . Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 1996; 274:249–252.
Petreaca RC, Chiu HC, Eckelhoefer HA, Chuang C, Xu L, Nugent CI . Chromosome end protection plasticity revealed by Stn1p and Ten1p bypass of Cdc13p. Nat Cell Biol 2006; 8:748–755.
Petreaca RC, Chiu HC, Nugent CI . The role of Stn1p in Saccharomyces cerevisiae telomere capping can be separated from its interaction with Cdc13p. Genetics 2007; 177:1459–1474.
Puglisi A, Bianchi A, Lemmens L, Damay P, Shore D . Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition. EMBO J 2008; 27:2328–2339.
Gilson E, Geli V . How telomeres are replicated. Nat Rev Mol Cell Biol 2007; 8:825–838.
Sampathi S, Chai W . Telomere replication: poised but puzzling. J Cell Mol Med 2011; 15:3–13.
Miller KM, Rog O, Cooper JP . Semi-conservative DNA replication through telomeres requires Taz1. Nature 2006; 440:824–828.
Ye J, Lenain C, Bauwens S, et al. TRF2 and apollo cooperate with topoisomerase 2alpha to protect human telomeres from replicative damage. Cell 2010; 142:230–242.
Sfeir A, Kosiyatrakul ST, Hockemeyer D, et al. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell 2009; 138:90–103.
Martinez P, Thanasoula M, Munoz P, et al. Increased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice. Genes Dev 2009; 23:2060–2075.
Badie S, Escandell JM, Bouwman P, et al. BRCA2 acts as a RAD51 loader to facilitate telomere replication and capping. Nat Struct Mol Biol 2010; 17:1461–1469.
Crabbe L, Verdun RE, Haggblom CI, Karlseder J . Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science 2004; 306:1951–1953.
Chai W, Du Q, Shay JW, Wright WE . Human telomeres have different overhang sizes at leading versus lagging strands. Mol Cell 2006; 21:427–435.
Saharia A, Teasley DC, Duxin JP, Dao B, Chiappinelli KB, Stewart SA . FEN1 ensures telomere stability by facilitating replication fork re-initiation. J Biol Chem 2010; 285:27057–27066.
Zou L, Elledge SJ . Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003; 300:1542–1548.
Zou L . Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response. Genes Dev 2007; 21:879–885.
Guo X, Deng Y, Lin Y, et al. Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis. EMBO J 2007; 26:4709–4719.
Denchi EL, de Lange T . Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature 2007; 448:1068–1071.
Flynn RL, Centore RC, O'Sullivan RJ, et al. TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA. Nature 2011; 471:532–536.
Wu L, Multani AS, He H, et al. Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres. Cell 2006; 126:49–62.
He H, Wang Y, Guo X, et al. Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita. Mol Cell Biol 2009; 29:229–240.
Zhao Y, Sfeir AJ, Zou Y, et al. Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells. Cell 2009; 138:463–475.
Chai W, Shay JW, Wright WE . Human telomeres maintain their overhang length at senescence. Mol Cell Biol 2005; 25:2158–2168.
Goulian M, Heard CJ, Grimm SL . Purification and properties of an accessory protein for DNA polymerase alpha/primase. J Biol Chem 1990; 265:13221–13230.
Goulian M, Heard CJ . The mechanism of action of an accessory protein for DNA polymerase alpha/primase. J Biol Chem 1990; 265:13231–13239.
Gu P, Min JN, Wang Y, et al. CTC1 deletion results in defective telomere replication, leading to catastrophic telomere loss and stem cell exhaustion. EMBO J 2012; 31:2309–2321.
Zhu W, Ukomadu C, Jha S, et al. Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. Genes Dev 2007; 21:2288–2299.
Chattopadhyay S, Bielinsky AK . Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication. Mol Biol Cell 2007; 18:4085–4095.
Ricke RM, Bielinsky AK . Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha. Mol Cell 2004; 16:173–185.
Verdun RE, Karlseder J . The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres. Cell 2006; 127:709–720.
Nakaoka H, Nishiyama A, Saito M, Ishikawa F . Xenopus laevis Ctc1-Stn1-Ten1 (xCST) complex is involved in priming DNA synthesis on single-stranded DNA template in Xenopus egg extract. J Biol Chem 2012; 287:619–627.
Hockemeyer D, Sfeir AJ, Shay JW, Wright WE, de Lange T . POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end. EMBO J 2005; 24:2667–2678.
Hockemeyer D, Palm W, Wang RC, Couto SS, de Lange T . Engineered telomere degradation models dyskeratosis congenita. Genes Dev 2008; 22:1773–1785.
Petermann E, Helleday T . Pathways of mammalian replication fork restart. Nat Rev Mol Cell Biol 2010; 11:683–687.
Allen C, Ashley AK, Hromas R, Nickoloff JA . More forks on the road to replication stress recovery. J Mol Cell Biol 2011; 3:4–12.
Vannier J-BP-K, Visnja, Petalcorin MIR, Hao D, Boulton SJ . RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity. Cell 2012; 149:795–806.
Dai X, Huang C, Chai W . CDK1 differentially regulates G-overhang generation at leading- and lagging- strand telomeres in telomerase negative cells in the G2 phase. Cell Cycle 2012; 11:3079–3086.
Sampathi S, Bhusari A, Shen B, Chai W . Human Flap Endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance. J Biol Chem 2009; 284:3682–3690.
Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 1995; 92:9363–9367.
Bailey SM, Cornforth MN, Kurimasa A, Chen DJ, Goodwin EH . Strand-specific postreplicative processing of mammalian telomeres. Science 2001; 293:2462–2465.
Acknowledgements
We thank S Chang for communicating results prior to publication. We also thank Z Songyang (Baylor College of Medicine, USA) for pBabe-FLAG-Stn1. This work was supported by NIH R15GM099008 and American Cancer Society Institutional Research Grant IRG-77-003-32 to WC.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Anti-Stn1 antibody recognizes Stn1 protein. (PDF 417 kb)
Supplementary information, Figure S2
No increase in T-SCE or signal free ends (SFE) after Stn1 depletion in HeLa 1.2.11. (PDF 212 kb)
Supplementary information, Figure S3
Separation of replicated and unreplicated telomeres using CsCl ultracentrifugation. (PDF 934 kb)
Supplementary information, Figure S4
Growth curve of BJ and BJ/E6/E7 expressing shLuc and shStn1. (PDF 193 kb)
Supplementary information, Figure S5
Short STELA products resulted largely from telorettes ligating at telomere ends. (PDF 586 kb)
Supplementary information, Figure S6
Co-IP of polα and Stn1 throughout the cell cycle. (PDF 293 kb)
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Huang, C., Dai, X. & Chai, W. Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in. Cell Res 22, 1681–1695 (2012). https://doi.org/10.1038/cr.2012.132
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DOI: https://doi.org/10.1038/cr.2012.132
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