Skip to main content
SpringerLink
Log in

Mrc1, a non-essential DNA replication protein, is required for telomere end protection following loss of capping by Cdc13, Yku or telomerase

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

Proteins involved in telomere end protection have previously been identified. In Saccharomyces cerevisiae, Cdc13, Yku and telomerase, mainly, prevent telomere uncapping, thus providing telomere stability and avoiding degradation and death by senescence. Here, we report that in the absence of Mrc1, a component of the replication forks, telomeres of cdc13 or yku70 mutants exhibited increased degradation, while telomerase-negative cells displayed accelerated senescence. Moreover, deletion of MRC1 increased the single-strandedness of the telomeres in cdc13-1 and yku70Δ mutant strains. An mrc1 deletion strain also exhibited slight but stable telomere shortening compared to a wild-type strain. Loss of Mrc1’s checkpoint function alone did not provoke synthetic growth defects in combination with the cdc13-1 mutation. Combinations between the cdc13-1 mutation and deletion of either TOF1 or PSY2, coding for proteins physically interacting with Mrc1, also resulted in a synthetic growth defect. Thus, the present data suggest that non-essential components of the DNA replication machinery, such as Mrc1 and Tof1, may have a role in telomere stability in addition to their role in fork progression.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Alcasabas AA, Osborn AJ, Bachant J, Hu F, Werler PJ, Bousset K, Furuya K, Diffley JF, Carr AM, Elledge SJ (2001) Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat Cell Biol 3:958–965

    Article  PubMed  CAS  Google Scholar 

  • Archambault V, Ikui AE, Drapkin BJ, Cross FR (2005) Disruption of mechanisms that prevent rereplication triggers a DNA damage response. Mol Cell Biol 25:6707–6721

    Article  PubMed  CAS  Google Scholar 

  • Aroya SB, Kupiec M (2005) The Elg1 replication factor C-like complex: a novel guardian of genome stability. DNA Rep 4:409–417

    Article  Google Scholar 

  • Askree SH, Yehuda T, Smolikov S, Gurevich R, Hawk J, Coker C, Krauskopf A, Kupiec M, McEachern MJ (2004) A genome-wide screen for Saccharomyces cerevisiae deletion mutants that affect telomere length. Proc Natl Acad Sci USA 101:8658–8663

    Article  PubMed  CAS  Google Scholar 

  • Bertuch AA, Lundblad V (2003) The Ku heterodimer performs separable activities at double-strand breaks and chromosome termini. Mol Cell Biol 8202–8215

  • Biggins S, Bhalla N, Chang A, Smith DL, Murray AW (2001) Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae. Genetics 159:453–470

    PubMed  CAS  Google Scholar 

  • Blackburn EH (2001) Switching and signaling at the telomere. Cell 106:661–673

    Article  PubMed  CAS  Google Scholar 

  • Boulton SJ, Jackson SP (1996) Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucl Acids Res 24:4639–4648

    Article  PubMed  CAS  Google Scholar 

  • Boulton SJ, Jackson SP (1998) Components of the Ku-dependent non-homologous end-joining pathway are involved in telomere length maintenance and telomeric silencing. EMBO J 6:1819–1828

    Article  Google Scholar 

  • Calzada A, Hodgson B, Kanemaki M, Bueno A, Labib K (2005) Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev 19:1905–1919

    Article  PubMed  CAS  Google Scholar 

  • Chakhparonian M, Wellinger RJ (2003) Telomere maintenance and DNA replication: how closely are these two connected? Trends Genet 19:439–444

    Article  PubMed  CAS  Google Scholar 

  • Chamankhah M, Fontanie T, Xiao W (2000) The Saccharomyces cerevisiae mre11 (ts) allele confers a separation of DNA repair and telomere maintenance functions. Genetics 155:569–576

    PubMed  CAS  Google Scholar 

  • Corda Y, Schramke V, Longhese MP, Smokvina T, Paciotti V, Brevet V, Gilson E, Géli V (1999) Interaction between Set1p and checkpoint protein Mec3p in DNA repair and telomere functions. Nat Genet 21:204–208

    Article  PubMed  CAS  Google Scholar 

  • Daniel JA, Keyes BE, Ng YP, Freeman CO, Burke DJ (2006) Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae. Genetics 172:53–65

    Article  PubMed  CAS  Google Scholar 

  • Davierwala A et al (2005) The synthetic genetic interaction spectrum of essential genes. Nat Genet 37:1147–1152

    Article  PubMed  CAS  Google Scholar 

  • DuBois ML, Haimberger ZW, McIntosh MW, Gottschling DE (2002) A quantitative assay for telomere protection in Saccharomyces cerevisiae. Genetics 161:995–1013

    PubMed  CAS  Google Scholar 

  • Ferreira MG, Miller KM, Cooper JP (2004) Indecent exposure: when telomeres become uncapped. Mol Cell 13:7–18

    Article  PubMed  CAS  Google Scholar 

  • Fisher TS, Zakian VA (2005) Ku: a multifunctional protein involved in telomere maintenance. DNA Rep 4:1215–1226

    Article  CAS  Google Scholar 

  • Foss EJ (2001) Tof1p regulates DNA damage responses during S phase in Saccharomyces cerevisiae. Genetics 157:567–577

    PubMed  CAS  Google Scholar 

  • Fouché N, Özgür S, Roy D, Griffith JD (2006) Replication fork regression in repetitive DNAs. Nucl Acid Res 34:6044–6050

    Article  Google Scholar 

  • Garvik B, Carson M, Hartwell L (1995) Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol Cell Biol 15:6128–6138

    PubMed  CAS  Google Scholar 

  • Gatbonton T, Imbesi M, Nelson M, Akey JM, Ruderfer DM, Kruglyak L, Simon JA, Bedalov A (2006) Telomere length as a quantitative trait: genome-wide survey and genetic mapping of telomere length-control genes in yeast. PLoS Genet 2:0304–0315

    CAS  Google Scholar 

  • Gingras AC, Caballero M, Zarske M, Sanchez A, Hazbun TR, Fields S, Sonenberg N, Hafen E, Raught B, Aebersold R (2005) A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol Cell Proteomics 4:1725–1740

    Article  PubMed  CAS  Google Scholar 

  • Grandin N, Charbonneau M (2003) The Rad51 pathway of telomerase-independent maintenance of telomeres can amplify TG1–3 sequences in yku and cdc13 mutants of Saccharomyces cerevisiae. Mol Cell Biol 23:3721–3734

    Article  PubMed  CAS  Google Scholar 

  • Grandin N, Reed SI, Charbonneau M (1997) Stn1, a new Saccharomyces cerevisiae protein, is implicated in telomere size regulation in association with Cdc13. Genes Dev 11:512–527

    PubMed  CAS  Google Scholar 

  • Grandin N, Bailly A, Charbonneau M (2005) Activation of Mrc1, a mediator of the replication checkpoint, by telomere erosion. Biol Cell 97:799–814

    Article  PubMed  CAS  Google Scholar 

  • Gravel S, Larrivée M, Labrecque P, Wellinger RJ (1998) Yeast Ku as a regulator of chromosomal DNA end structure. Science 280:741–744

    Article  PubMed  CAS  Google Scholar 

  • Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T (1999) Mammalian telomeres end in a large duplex loop. Cell 97:503–514

    Article  PubMed  CAS  Google Scholar 

  • Grossi S, Puglisi A, Dmitriev PV, Lopes M, Shore D (2004) Pol12, the B subunit of DNA polymerase α, functions in both telomere capping and length regulation. Genes Dev 18:992–1006

    Article  PubMed  CAS  Google Scholar 

  • Hackett JA, Feldser DM, Greider CW (2001) Telomere dysfunction increases mutation rate and genomic instability. Cell 106:275–286

    Article  PubMed  CAS  Google Scholar 

  • Hansay D, Chin JK, Xia G, Oshiro G, Winzeler EA, Romesberg FE (2002) Previously uncharacterized genes in the UV- and MMS-induced DNA damage response in yeast. Proc Natl Acad Sci USA 99:10605–10610

    Article  Google Scholar 

  • Hu F, Alcasabas AA, Elledge SJ (2001) Asf1 links Rad53 to control of chromatin assembly. Genes Dev 15:1061–1066

    Article  PubMed  CAS  Google Scholar 

  • Ivessa AS, Zhou JQ, Schulz VP, Monson EK, Zakian VA (2002) Saccharomyces Rrm3p, a 5’ to 3’ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA. Genes Dev 16:1383–1396

    Article  PubMed  CAS  Google Scholar 

  • Ivessa AS, Lenzmeier BA, Bessler JB, Goudsouzian LK, Schnakenberg SL, Zakian VA (2003) The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes. Mol Cell 12:1525–1536

    Article  PubMed  CAS  Google Scholar 

  • Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424:1078–1083

    Article  PubMed  CAS  Google Scholar 

  • Keogh MC et al (2006) A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery. Nature 439:497–501

    Article  PubMed  CAS  Google Scholar 

  • Kim ST, Lim DS, Canman CE, Kastan MB (1999) Substrate specificities and identification of putative substrates of ATM kinase family members. J Biol Chem 274:37538–37543

    Article  PubMed  CAS  Google Scholar 

  • Krogan NJ et al (2003) A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1. Mol Cell 12:1565–1576

    Article  PubMed  CAS  Google Scholar 

  • Kumagai A, Dunphy WG (2000) Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol Cell 6:839–849

    Article  PubMed  CAS  Google Scholar 

  • de Lange T (2004) T-loops and the origin of telomeres. Nat Rev Mol Cell Biol 5:323–329

    Article  PubMed  Google Scholar 

  • Lendvay TS, Morris DK, Sah J, Balasubramanian B, Lundblad V (1996) Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes. Genetics 144:1399–1412

    PubMed  CAS  Google Scholar 

  • Lin JJ, Zakian VA (1996) The Saccharomyces CDC13 protein is a single-strand TG1–3 telomeric DNA-binding protein in vitro that affects telomere behavior in vivo. Proc Natl Acad Sci USA 93:13760–13765

    Article  PubMed  CAS  Google Scholar 

  • Lingner J, Hughes TR, Shevchenko A, Mann M, Lundblad V, Cech TR (1997) Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276:561–567

    Article  PubMed  CAS  Google Scholar 

  • Loeillet S, Palancade B, Cartron M, Thierry A, Richard GF, Dujon B, Doye V, Nicolas A (2005) Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast. DNA Rep 4:459–468

    Article  CAS  Google Scholar 

  • Longhese MP, Paciotti V, Neecke H, Lucchini G (2000) Checkpoint proteins influence telomeric silencing and length maintenance in budding yeast. Genetics 155:1577–1591

    PubMed  CAS  Google Scholar 

  • Louis EJ, Borts RH (1995) A complete set of marked telomeres in Saccharomyces cerevisiae for physical mapping and cloning. Genetics 139:125–136

    PubMed  CAS  Google Scholar 

  • Lundblad V (2002) Telomere maintenance without telomerase. Oncogene 21:522–531

    Article  PubMed  CAS  Google Scholar 

  • Lundblad V, Blackburn EH (1993) An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell 73:347–360

    Article  PubMed  CAS  Google Scholar 

  • Lundblad V, Szostak JW (1989) A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57:633–643

    Article  PubMed  CAS  Google Scholar 

  • Lustig AJ (2001) Cdc13 subcomplexes regulate multiple telomere functions. Nat Struct Biol 8:297–299

    Article  PubMed  CAS  Google Scholar 

  • Lustig AJ, Petes TD (1986) Identification of yeast mutants with altered telomere structure. Proc Natl Acad Sci USA 83:1398–1402

    Article  PubMed  CAS  Google Scholar 

  • Mallory JC, Bashkirov VI, Trujillo KM, Solinger JA, Dominska M, Sung P, Heyer WD, Petes TD (2003) Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae. DNA Rep 2:1041–1064

    Article  CAS  Google Scholar 

  • Maringele L, Lydall D (2002) EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Δ mutants. Genes Dev 16:1919–1933

    Article  PubMed  CAS  Google Scholar 

  • Matheos D, Ruiz MT, Price GB, Zannis-Hadjopoulos M (2002) Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication. Biochim Biophys Acta 1578:59–72

    PubMed  CAS  Google Scholar 

  • Mohanty BK, Bairwa NK, Bastia D (2006) The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 103:897–902

    Article  PubMed  CAS  Google Scholar 

  • Nugent CI, Hughes TR, Lue NF, Lundblad V (1996) Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 274:249–252

    Article  PubMed  CAS  Google Scholar 

  • Nugent CI, Bosco G, Ross LO, Evans SK, Salinger AP, Moore JK, Haber JE, Lundblad V (1998) Telomere maintenance is dependent on activities required for end repair of double-strand breaks. Curr Biol 8:657–660

    Article  PubMed  CAS  Google Scholar 

  • O’Neill BM, Hanway D, Winzeler EA, Romesberg FE (2004) Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response. Nucl Acids Res 32:6519–6530

    Article  PubMed  CAS  Google Scholar 

  • Osborn AJ, Elledge SJ (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev 17:1755–1767

    Article  PubMed  CAS  Google Scholar 

  • Pan X, Ye P, Yuan DS, Wang X, Bader JS, Boeke JD (2006) A DNA integrity network in the yeast Saccharomyces cerevisiae. Cell 124:1069–1081

    Article  PubMed  CAS  Google Scholar 

  • Peterson SE, Stellwagen AE, Diede SJ, Singer MS, Haimberger ZW, Johnson CO, Tzoneva M, Gottschling DE (2001) The function of a stem-loop in telomerase RNA is linked to the DNA repair protein Ku. Nat Genet 27:64–67

    PubMed  CAS  Google Scholar 

  • Polotnianka RM, Li J, Lustig AJ (1998) The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities. Curr Biol 8:831–834

    Article  PubMed  CAS  Google Scholar 

  • Qi H, Zakian VA (2000) The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein. Genes Dev 14:1777–1788

    PubMed  CAS  Google Scholar 

  • Singer MS, Gottschling DE (1994) TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266:404–409

    Article  PubMed  CAS  Google Scholar 

  • Smogorzewska A, de Lange T (2004) Regulation of telomerase by telomeric proteins. Ann Rev Biochem 73:177–208

    Article  PubMed  CAS  Google Scholar 

  • Smolikov S, Mazor Y, Krauskopf A (2004) ELG1, a regulator of genome stability, has a role in telomere length regulation and in silencing. Proc Natl Acad Sci USA 101:1656–1661

    Article  PubMed  CAS  Google Scholar 

  • Stellwagen AE, Haimberger ZW, Veatch JR, Gottschling DE (2003) Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev 17:2384–2395

    Article  PubMed  CAS  Google Scholar 

  • Suter B, Tong A, Chang M, Yu L, Brown GW, Boone C, Rine J (2004) The origin recognition complex links replication, sister chromatid cohesion and transcriptional silencing in Saccharomyces cerevisiae. Genetics 167:579–591

    Article  PubMed  CAS  Google Scholar 

  • Symington LS (2002) Role of RAD52 epistastis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 66:630–670

    Article  PubMed  CAS  Google Scholar 

  • Szyjka SJ, Viggiani CJ, Aparicio OM (2005) Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae. Mol Cell 19:691–697

    Article  PubMed  CAS  Google Scholar 

  • Tanaka K, Russell P (2001) Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1. Nat Cell Biol 3:966–972

    Article  PubMed  CAS  Google Scholar 

  • Teo SH, Jackson SP (2001) Telomerase subunit overexpression suppresses telomere-specific checkpoint activation in the yeast yku80 mutant. EMBO Rep 2:197–202

    Article  PubMed  CAS  Google Scholar 

  • Tong AH et al (2004) Global mapping of the yeast genetic interaction network. Science 303:808–813

    Article  PubMed  CAS  Google Scholar 

  • Tourrière H, Versini G, Cordon-Preciado V, Alabert C, Pasero P (2005) Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol Cell 19:699–706

    Article  PubMed  Google Scholar 

  • Uetz P et al (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403:601–603

    Article  Google Scholar 

  • Wellinger RJ, Wolf AJ., Zakian VA (1993) Saccharomyces telomeres acquire single-strand TG1–3 tails late in S phase. Cell 72:51–60

    Article  PubMed  CAS  Google Scholar 

  • Wu HI, Brown JA, Dorie MJ, Lazzeroni L, Brown JM (2004) Genome-wide identification of genes conferring resistance to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. Cancer Res 64:3940–3948

    Article  PubMed  CAS  Google Scholar 

  • Xu H, Boone C, Klein HL (2004) Mrc1 is required for sister chromatid cohesion to aid in recombination repair of spontaneous damage. Mol Cell Biol 24:7082–7090

    Article  PubMed  CAS  Google Scholar 

  • Zegerman P, Diffley JFX (2003) Lessons in how to hold a fork. Nat Struct Biol 10:778–779

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Stephen Elledge, Leland Hartwell, Dan Gottschling and Wei Xiao for the gifts of strains and plasmids. This work was supported by grants from the “Association pour la Recherche contre le Cancer”, the INCa (“Institut National du Cancer”; EPIPRO program to Saadi Kochblin and Eric Gilson) and the “Comité Départemental de la Savoie de la Ligue Nationale contre le Cancer”.

Author information

Authors and Affiliations

  1. UMR CNRS no 5161, Ecole Normale Supérieure de Lyon, IFR128 BioSciences Gerland, 46, allée d’Italie, 69364, Lyon, France

    Nathalie Grandin & Michel Charbonneau

Authors
  1. Nathalie Grandin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michel Charbonneau
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michel Charbonneau.

Additional information

Communicated by S. Hohmann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Grandin, N., Charbonneau, M. Mrc1, a non-essential DNA replication protein, is required for telomere end protection following loss of capping by Cdc13, Yku or telomerase. Mol Genet Genomics 277, 685–699 (2007). https://doi.org/10.1007/s00438-007-0218-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-007-0218-0

Keywords

Search

Navigation