Advertisement

FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation*

  • Abhishek Saharia
    Affiliations
    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Daniel C. Teasley
    Affiliations
    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Julien P. Duxin
    Affiliations
    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Benjamin Dao
    Affiliations
    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Katherine B. Chiappinelli
    Affiliations
    Division of Endocrine and Oncologic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Sheila A. Stewart
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8228, St. Louis, MO 63110. Tel.: 314-362-7437; Fax: 314-362-7463
    Affiliations
    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

    Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by the Children's Discovery Institute (to S. A. S.) and Cellular, Biochemical, and Molecular Sciences Predoctoral Training Grant T32 GM007067-35 (to D. C. T.).
Open AccessPublished:June 15, 2010DOI:https://doi.org/10.1074/jbc.M110.112276
      Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.

      Introduction

      High fidelity DNA replication is critical for genome stability and continued cellular proliferation. Given the importance of high fidelity DNA replication to genomic stability, it is not surprising that numerous redundant mechanisms of DNA replication exist. Inherited syndromes in which DNA replication/repair proteins are mutated or lost but overall DNA replication continues relatively unabated (
      • Wu L.
      • Hickson I.D.
      ,
      • Sidorova J.M.
      ,
      • Singh D.K.
      • Ahn B.
      • Bohr V.A.
      ) best illustrate the compensatory nature of these mechanisms. However, in some cases this compensation is incomplete, and thus, patients with these mutations manifest replication defects and genomic instability (
      • Sidorova J.M.
      ).
      Deficiencies in various DNA replication/repair mechanisms become particularly detrimental in highly repetitive DNA sequences that present unique challenges to the DNA replication machinery (
      • Gilson E.
      • Géli V.
      ,
      • Verdun R.E.
      • Karlseder J.
      ). For example, triplet repeats can lead to replication fork slippage, resulting in deleterious expansions and deletions (
      • Kovtun I.V.
      • McMurray C.T.
      ). Similarly, replication fork pausing and stalling occur within telomeric repeats (
      • Ohki R.
      • Ishikawa F.
      ,
      • Makovets S.
      • Herskowitz I.
      • Blackburn E.H.
      ,
      • Verdun R.E.
      • Karlseder J.
      ,
      • Khadaroo B.
      • Teixeira M.T.
      • Luciano P.
      • Eckert-Boulet N.
      • Germann S.M.
      • Simon M.N.
      • Gallina I.
      • Abdallah P.
      • Gilson E.
      • Géli V.
      • Lisby M.
      ), and telomeres were recently identified as fragile sites (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ,
      • Martínez P.
      • Thanasoula M.
      • Muñoz P.
      • Liao C.
      • Tejera A.
      • McNees C.
      • Flores J.M.
      • Fernández-Capetillo O.
      • Tarsounas M.
      • Blasco M.A.
      ). Because fragile sites are thought to arise in response to replication stress, these data support the hypothesis that telomeric DNA presents a challenging template for the DNA replication machinery that requires the actions of specialized replication complexes, including a replication fork re-initiation complex (
      • Gilson E.
      • Géli V.
      ,
      • Parkinson G.N.
      • Lee M.P.
      • Neidle S.
      ,
      • Maizels N.
      ). Recent work has shown that telomeres are highly sensitive to the loss of DNA replication/repair proteins shown to localize to stalled replication forks, including the Werner (WRN)
      The abbreviations used are: WRN
      Werner
      FEN1
      Flap endonuclease 1
      STL
      sister telomere loss
      GEN
      gap endonuclease
      CO-FISH
      orientation-FISH
      hWT
      human WT
      mWT
      mutant WT
      mED
      mutant E160D
      DA
      D181A
      HJ
      Holliday junction
      PCNA
      proliferating cell nuclear antigen
      BrdU
      5-bromo-2-deoxyuridine
      BLM
      Bloom
      PNA
      peptide nucleic acid.
      and Flap endonuclease 1 (FEN1) proteins (
      • Sharma S.
      • Otterlei M.
      • Sommers J.A.
      • Driscoll H.C.
      • Dianov G.L.
      • Kao H.I.
      • Bambara R.A.
      • Brosh Jr., R.M.
      ). Indeed, cells from WRN patients display overt telomere dysfunction, whereas only minor defects in genomic replication are observed (
      • Sidorova J.M.
      ,
      • Crabbe L.
      • Verdun R.E.
      • Haggblom C.I.
      • Karlseder J.
      ,
      • Crabbe L.
      • Jauch A.
      • Naeger C.M.
      • Holtgreve-Grez H.
      • Karlseder J.
      ), suggesting that other proteins compensate for WRN throughout the genome but are insufficient at the telomere.
      DNA replication mechanisms at the telomere are coordinated by the six-protein Shelterin complex (including TRF1, TRF2, TIN2, POT1, RAP1, and TPP1) (
      • Gilson E.
      • Géli V.
      ,
      • Verdun R.E.
      • Karlseder J.
      ,
      • de Lange T.
      ). For example, TRF2 interacts with and modulates the activities of numerous DNA replication and repair proteins (
      • de Lange T.
      ). These interactions include TRF2 binding to the WRN and BLM helicases, which stimulates their activity in vitro, suggesting that TRF2 recruits them to replicate or repair telomeric DNA (
      • Opresko P.L.
      • von Kobbe C.
      • Laine J.P.
      • Harrigan J.
      • Hickson I.D.
      • Bohr V.A.
      ). In Schizosaccharomyces pombe, the TRF1/2 homolog Taz1 is essential for DNA replication through the telomeres (
      • Miller K.M.
      • Rog O.
      • Cooper J.P.
      ). Upon Taz1 deletion, replication forks stall within telomeric repeats, and telomeres are rapidly lost (
      • Miller K.M.
      • Rog O.
      • Cooper J.P.
      ). TRF1 plays a similar role in mammalian cells (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ,
      • Martínez P.
      • Thanasoula M.
      • Muñoz P.
      • Liao C.
      • Tejera A.
      • McNees C.
      • Flores J.M.
      • Fernández-Capetillo O.
      • Tarsounas M.
      • Blasco M.A.
      ). After deletion of TRF1, stalled replication forks accumulate within the telomeric repeats, resulting in a replication stress response characterized by an ATR (ataxia telangiectasia mutated (ATM)- and Rad3-related)-dependent DNA damage response and expression of fragile sites within telomeric DNA (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ,
      • Martínez P.
      • Thanasoula M.
      • Muñoz P.
      • Liao C.
      • Tejera A.
      • McNees C.
      • Flores J.M.
      • Fernández-Capetillo O.
      • Tarsounas M.
      • Blasco M.A.
      ). Together, these data underscore the importance of the coordinated actions of the Shelterin components and the DNA replication and repair machinery to efficiently complete telomere replication.
      FEN1 is a structure-specific endonuclease that plays an important role in DNA metabolism. FEN1 participates in Okazaki fragment processing during lagging strand DNA replication (
      • Li X.
      • Li J.
      • Harrington J.
      • Lieber M.R.
      • Burgers P.M.
      ) and is important for several DNA repair processes (
      • Liu Y.
      • Kao H.I.
      • Bambara R.A.
      ,
      • Shen B.
      • Singh P.
      • Liu R.
      • Qiu J.
      • Zheng L.
      • Finger L.D.
      • Alas S.
      ). FEN1 co-localizes to stalled replication forks where it interacts with the RecQ helicase, WRN, and is postulated to re-initiate stalled DNA replication forks (
      • Sharma S.
      • Otterlei M.
      • Sommers J.A.
      • Driscoll H.C.
      • Dianov G.L.
      • Kao H.I.
      • Bambara R.A.
      • Brosh Jr., R.M.
      ,
      • Zheng L.
      • Zhou M.
      • Chai Q.
      • Parrish J.
      • Xue D.
      • Patrick S.M.
      • Turchi J.J.
      • Yannone S.M.
      • Chen D.
      • Shen B.
      ). Recently, we demonstrated that FEN1 is vital for telomere stability (
      • Saharia A.
      • Guittat L.
      • Crocker S.
      • Lim A.
      • Steffen M.
      • Kulkarni S.
      • Stewart S.A.
      ). Indeed, FEN1 depletion in telomerase-deficient cells leads to a DNA damage response at telomeres and telomere dysfunction characterized by loss of lagging strand-replicated sister telomeres (STLs) (
      • Saharia A.
      • Guittat L.
      • Crocker S.
      • Lim A.
      • Steffen M.
      • Kulkarni S.
      • Stewart S.A.
      ,
      • Saharia A.
      • Stewart S.A.
      ). Furthermore, genetic rescue experiments demonstrate that the nuclease activity and the C-terminal WRN-interacting domain of FEN1 are important for telomere stability (
      • Saharia A.
      • Guittat L.
      • Crocker S.
      • Lim A.
      • Steffen M.
      • Kulkarni S.
      • Stewart S.A.
      ).
      The above findings prompted us to investigate how FEN1 contributes to telomere stability. Here, for the first time we demonstrate that FEN1 promotes efficient re-initiation of stalled replication forks. The C-terminal domain of FEN1 and its gap endonuclease (GEN) activity are critical for its ability to re-initiate stalled replication forks. However, FEN1 depletion does not affect progression through S phase or SV40 large T antigen-dependent in vitro DNA replication of non-repetitive sequences. Instead, FEN1 depletion leads to replicative stress within telomeric sequences as evidenced by expression of fragile sites. Finally, we demonstrate that the PCNA-interacting domain of FEN1 is dispensable for its telomere function and that the GEN activity is critical for its ability to prevent STLs. We propose that FEN1 maintains stable telomeres through efficient re-initiation of stalled replication forks that occur in the G-rich telomere, ensuring high fidelity telomere replication.

      DISCUSSION

      Telomeres perform a critical cellular function by distinguishing the chromosome end from a bona fide double-stranded DNA break. As such, mechanisms that modify the activities of DNA repair and replication proteins, presumably through interaction with the Shelterin complex, have evolved to protect the telomere and ensure its faithful replication. The need for telomere-specific replication mechanisms is likely due to the nature of the telomeric DNA sequence, which presents a number of challenges to the DNA replication machinery (
      • Gilson E.
      • Géli V.
      ,
      • Verdun R.E.
      • Karlseder J.
      ). G-rich, repetitive, telomeric sequences have a high propensity to form secondary structures such as G-quadruplexes (G4) that impede the progressing replication fork, leading to the formation of stalled forks (
      • Gilson E.
      • Géli V.
      ,
      • Parkinson G.N.
      • Lee M.P.
      • Neidle S.
      ,
      • Maizels N.
      ). Indeed, telomeres were recently identified as fragile sites (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ), and several reports have indicated pausing or stalling of replication forks within telomeres (
      • Makovets S.
      • Herskowitz I.
      • Blackburn E.H.
      ,
      • Verdun R.E.
      • Karlseder J.
      ,
      • Khadaroo B.
      • Teixeira M.T.
      • Luciano P.
      • Eckert-Boulet N.
      • Germann S.M.
      • Simon M.N.
      • Gallina I.
      • Abdallah P.
      • Gilson E.
      • Géli V.
      • Lisby M.
      ,
      • Ivessa A.S.
      • Zhou J.Q.
      • Schulz V.P.
      • Monson E.K.
      • Zakian V.A.
      ). Additionally, telomere replication is primarily initiated by the most centromere-distal origin of replication and continues unidirectionally toward the end of the telomere (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ). If a replication fork stalls within the telomere and is not re-initiated, the absence of a converging replication fork would result in telomere loss. Therefore, mechanisms that facilitate replication fork movement through the telomere are critical to high fidelity telomere replication.
      The importance of the Shelterin complex to telomere replication is underscored by several studies. For example, Taz1 in S. pombe and TRF1 in mice are required for efficient telomere replication. Loss of Taz1 results in replication fork stalling throughout telomeric sequences (
      • Miller K.M.
      • Rog O.
      • Cooper J.P.
      ), whereas loss of TRF1 leads to expression of fragile telomeres (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ). The ability of telomere-binding proteins to facilitate replication fork progression through the telomere is postulated to require recruitment of specialized proteins (
      • Gilson E.
      • Géli V.
      ,
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ). For example, TRF1 and TRF2 interact with and stimulate the RecQ helicases, BLM and WRN (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ,
      • Lillard-Wetherell K.
      • Machwe A.
      • Langland G.T.
      • Combs K.A.
      • Behbehani G.K.
      • Schonberg S.A.
      • German J.
      • Turchi J.J.
      • Orren D.K.
      • Groden J.
      ,
      • Opresko P.L.
      • Otterlei M.
      • Graakjaer J.
      • Bruheim P.
      • Dawut L.
      • Kølvraa S.
      • May A.
      • Seidman M.M.
      • Bohr V.A.
      ), suggesting that they recruit these proteins to enhance DNA replication and/or repair at the telomeres. Interestingly, a recent study demonstrated that TRF2 increases branch migration of Holliday Junction (HJ) intermediates, suggesting that this promotes the formation of chicken foot structures in the context of a stalled replication fork at telomeres (
      • Poulet A.
      • Buisson R.
      • Faivre-Moskalenko C.
      • Koelblen M.
      • Amiard S.
      • Montel F.
      • Cuesta-Lopez S.
      • Bornet O.
      • Guerlesquin F.
      • Godet T.
      • Moukhtar J.
      • Argoul F.
      • Déclais A.C.
      • Lilley D.M.
      • Ip S.C.
      • West S.C.
      • Gilson E.
      • Giraud-Panis M.J.
      ). FEN1 also interacts with TRF2 (
      • Saharia A.
      • Guittat L.
      • Crocker S.
      • Lim A.
      • Steffen M.
      • Kulkarni S.
      • Stewart S.A.
      ,
      • Muftuoglu M.
      • Wong H.K.
      • Imam S.Z.
      • Wilson 3rd, D.M.
      • Bohr V.A.
      • Opresko P.L.
      ), and because FEN1 GEN activity is postulated to process chicken foot structures (
      • Zheng L.
      • Zhou M.
      • Chai Q.
      • Parrish J.
      • Xue D.
      • Patrick S.M.
      • Turchi J.J.
      • Yannone S.M.
      • Chen D.
      • Shen B.
      ,
      • Liu R.
      • Qiu J.
      • Finger L.D.
      • Zheng L.
      • Shen B.
      ), this raises the possibility that TRF2 engages the RecQ helicase-FEN1 complex coordinately at the telomere to resolve stalled replication forks and enable their efficient restart.
      WRN participates in the re-initiation of stalled replication forks in vivo (
      • Dhillon K.K.
      • Sidorova J.
      • Saintigny Y.
      • Poot M.
      • Gollahon K.
      • Rabinovitch P.S.
      • Monnat Jr., R.J.
      ,
      • Sidorova J.M.
      • Li N.
      • Folch A.
      • Monnat Jr., R.J.
      ). Interestingly, FEN1 was shown to localize with WRN, raising the possibility that it contributes to replication fork restart (
      • Sharma S.
      • Otterlei M.
      • Sommers J.A.
      • Driscoll H.C.
      • Dianov G.L.
      • Kao H.I.
      • Bambara R.A.
      • Brosh Jr., R.M.
      ). Furthermore, FEN1 and WRN process branch migrating structures that resemble regressed replication forks in vitro (
      • Sharma S.
      • Otterlei M.
      • Sommers J.A.
      • Driscoll H.C.
      • Dianov G.L.
      • Kao H.I.
      • Bambara R.A.
      • Brosh Jr., R.M.
      ). The present study demonstrates for the first time that FEN1 functionally participates in the re-initiation of stalled replication forks in vivo. Together with previous work (
      • Nikolova T.
      • Christmann M.
      • Kaina B.
      ), this indicates that the FEN1 role in S phase is 2-fold; first, in Okazaki fragment processing during DNA replication and, second, in the re-initiation of stalled replication forks. FEN1 localizes to mammalian telomeres during S phase (
      • Verdun R.E.
      • Karlseder J.
      ,
      • Saharia A.
      • Guittat L.
      • Crocker S.
      • Lim A.
      • Steffen M.
      • Kulkarni S.
      • Stewart S.A.
      ), so it could be involved in one or both of the functions outlined above. However, given that the PCNA-interacting domain of FEN1 is dispensable for telomere stability, our data indicate that the role of FEN1 in Okazaki fragment processing is non-essential for telomere stability. This result indicates that either sufficient FEN1 remains in FEN1-depleted cells to support continued replication or that other nucleases such as DNA2 or EXO1, which can also process Okazaki fragments (
      • Moreau S.
      • Morgan E.A.
      • Symington L.S.
      ,
      • Kang H.Y.
      • Choi E.
      • Bae S.H.
      • Lee K.H.
      • Gim B.S.
      • Kim H.D.
      • Park C.
      • MacNeill S.A.
      • Seo Y.S.
      ,
      • Bae S.H.
      • Seo Y.S.
      ,
      • Ayyagari R.
      • Gomes X.V.
      • Gordenin D.A.
      • Burgers P.M.
      ,
      • Kao H.I.
      • Campbell J.L.
      • Bambara R.A.
      ), compensate for FEN1 loss during lagging strand DNA replication. However, these same nucleases are insufficient when replication forks stall within telomeric sequences. Indeed, we find that in the absence of the ability of FEN1 to re-initiate stalled replication forks, sister telomeres are lost despite the presence of other nucleases. Interestingly, other proteins involved in the re-initiation of stalled replication forks such as PARP1 and PARP2 have also been implicated in telomere maintenance (
      • Dantzer F.
      • Giraud-Panis M.J.
      • Jaco I.
      • Amé J.C.
      • Schultz I.
      • Blasco M.
      • Koering C.E.
      • Gilson E.
      • Ménissier-de Murcia J.
      • de Murcia G.
      • Schreiber V.
      ,
      • Ye J.Z.
      • de Lange T.
      ,
      • Bryant H.E.
      • Petermann E.
      • Schultz N.
      • Jemth A.S.
      • Loseva O.
      • Issaeva N.
      • Johansson F.
      • Fernandez S.
      • McGlynn P.
      • Helleday T.
      ), further indicating the importance of the re-initiation process for efficient telomere replication. An alternate hypothesis is that FEN1 is important for fork stabilization after hydroxyurea treatment. The assay we have conducted cannot differentiate between FEN1-dependent fork stabilization and fork re-initiation.
      Intriguingly, the C-terminal region of FEN1 is essential for its function at the telomere and also mediates its interaction with another RecQ helicase, BLM (
      • Sharma S.
      • Sommers J.A.
      • Gary R.K.
      • Friedrich-Heineken E.
      • Hübscher U.
      • Brosh Jr., R.M.
      ). Similar to WRN, BLM is able to unwind G4 DNA, is critical for the re-initiation of stalled replication forks, and has recently been suggested to be important for efficient telomere replication (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ,
      • Sengupta S.
      • Linke S.P.
      • Pedeux R.
      • Yang Q.
      • Farnsworth J.
      • Garfield S.H.
      • Valerie K.
      • Shay J.W.
      • Ellis N.A.
      • Wasylyk B.
      • Harris C.C.
      ,
      • Sun H.
      • Karow J.K.
      • Hickson I.D.
      • Maizels N.
      ,
      • Davies S.L.
      • North P.S.
      • Hickson I.D.
      ). This suggests that there is complicated interplay between WRN, BLM, and FEN1 at mammalian telomeres. Although the function of BLM at telomeres has not been well characterized, recent work suggests that it is important for repression of fragile telomeres (
      • Sfeir A.
      • Kosiyatrakul S.T.
      • Hockemeyer D.
      • MacRae S.L.
      • Karlseder J.
      • Schildkraut C.L.
      • de Lange T.
      ). Interestingly, FEN1 depletion also leads to an increase fragile telomere expression, raising the possibility that these proteins work as a complex to repress telomere fragility. Together, these data are consistent with the hypothesis that FEN1 and the RecQ helicases play an important role in the maintenance of stable telomeres through re-initiation of stalled replication forks.
      Here we demonstrate that FEN1 is important for efficient re-initiation of stalled replication forks in vivo. This function of FEN1 is dependent on its C-terminal domain and its GEN activity. However, despite the importance of FEN1 in re-initiation of stalled replication forks, FEN1 depletion in telomerase-positive cells did not affect S phase progression or SV40 Large T antigen-dependent in vitro DNA replication, suggesting that other nucleases compensate for FEN1-dependent replication function throughout the genome. However, these same proteins fail to compensate for FEN1 at the telomere. Indeed, FEN1 depletion leads to increased telomere fragility and lagging strand STLs. As with the re-initiation of stalled replication forks, both the FEN1 C terminus and GEN activity are essential for its function at telomeres, whereas its ability to interact with PCNA is dispensable. Collectively, these data demonstrate that FEN1 is necessary for efficient replication of telomeres, and we propose that FEN1 promotes replication fork re-initiation within telomeric sequences.

      Acknowledgments

      We are grateful to Dr. Ulrich Hübscher for providing the FEN1ΔP cDNA, Dr. Binghui Shen for providing the mWT and mED FEN1 cDNAs, and Dr. Fuyuki Ishikawa for providing the pSVO.11–2K plasmid. We are also grateful to Dr. Marc Wold, Dr. Peter Burgers, and members of the Stewart Laboratory for valuable discussions and to Ying Jie Lock, Ermira Pazolli, and Dr. Susana Gonzalo for critical reviews.

      REFERENCES

        • Wu L.
        • Hickson I.D.
        Mutat. Res. 2002; 509: 35-47
        • Sidorova J.M.
        DNA Repair. 2008; 7: 1776-1786
        • Singh D.K.
        • Ahn B.
        • Bohr V.A.
        Biogerontology. 2009; 10: 235-252
        • Gilson E.
        • Géli V.
        Nat. Rev. Mol. Cell Biol. 2007; 8: 825-838
        • Verdun R.E.
        • Karlseder J.
        Nature. 2007; 447: 924-931
        • Kovtun I.V.
        • McMurray C.T.
        Cell Res. 2008; 18: 198-213
        • Ohki R.
        • Ishikawa F.
        Nucleic Acids Res. 2004; 32: 1627-1637
        • Makovets S.
        • Herskowitz I.
        • Blackburn E.H.
        Mol. Cell. Biol. 2004; 24: 4019-4031
        • Verdun R.E.
        • Karlseder J.
        Cell. 2006; 127: 709-720
        • Khadaroo B.
        • Teixeira M.T.
        • Luciano P.
        • Eckert-Boulet N.
        • Germann S.M.
        • Simon M.N.
        • Gallina I.
        • Abdallah P.
        • Gilson E.
        • Géli V.
        • Lisby M.
        Nat. Cell Biol. 2009; 11: 980-987
        • Sfeir A.
        • Kosiyatrakul S.T.
        • Hockemeyer D.
        • MacRae S.L.
        • Karlseder J.
        • Schildkraut C.L.
        • de Lange T.
        Cell. 2009; 138: 90-103
        • Martínez P.
        • Thanasoula M.
        • Muñoz P.
        • Liao C.
        • Tejera A.
        • McNees C.
        • Flores J.M.
        • Fernández-Capetillo O.
        • Tarsounas M.
        • Blasco M.A.
        Genes Dev. 2009; 23: 2060-2075
        • Parkinson G.N.
        • Lee M.P.
        • Neidle S.
        Nature. 2002; 417: 876-880
        • Maizels N.
        Nat. Struct. Mol. Biol. 2006; 13: 1055-1059
        • Sharma S.
        • Otterlei M.
        • Sommers J.A.
        • Driscoll H.C.
        • Dianov G.L.
        • Kao H.I.
        • Bambara R.A.
        • Brosh Jr., R.M.
        Mol. Biol. Cell. 2004; 15: 734-750
        • Crabbe L.
        • Verdun R.E.
        • Haggblom C.I.
        • Karlseder J.
        Science. 2004; 306: 1951-1953
        • Crabbe L.
        • Jauch A.
        • Naeger C.M.
        • Holtgreve-Grez H.
        • Karlseder J.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2205-2210
        • de Lange T.
        Genes Dev. 2005; 19: 2100-2110
        • Opresko P.L.
        • von Kobbe C.
        • Laine J.P.
        • Harrigan J.
        • Hickson I.D.
        • Bohr V.A.
        J. Biol. Chem. 2002; 277: 41110-41119
        • Miller K.M.
        • Rog O.
        • Cooper J.P.
        Nature. 2006; 440: 824-828
        • Li X.
        • Li J.
        • Harrington J.
        • Lieber M.R.
        • Burgers P.M.
        J. Biol. Chem. 1995; 270: 22109-22112
        • Liu Y.
        • Kao H.I.
        • Bambara R.A.
        Annu. Rev. Biochem. 2004; 73: 589-615
        • Shen B.
        • Singh P.
        • Liu R.
        • Qiu J.
        • Zheng L.
        • Finger L.D.
        • Alas S.
        BioEssays. 2005; 27: 717-729
        • Zheng L.
        • Zhou M.
        • Chai Q.
        • Parrish J.
        • Xue D.
        • Patrick S.M.
        • Turchi J.J.
        • Yannone S.M.
        • Chen D.
        • Shen B.
        EMBO Rep. 2005; 6: 83-89
        • Saharia A.
        • Guittat L.
        • Crocker S.
        • Lim A.
        • Steffen M.
        • Kulkarni S.
        • Stewart S.A.
        Curr. Biol. 2008; 18: 496-500
        • Saharia A.
        • Stewart S.A.
        Oncogene. 2009; 28: 1162-1167
        • Stewart S.A.
        • Hahn W.C.
        • O'Connor B.F.
        • Banner E.N.
        • Lundberg A.S.
        • Modha P.
        • Mizuno H.
        • Brooks M.W.
        • Fleming M.
        • Zimonjic D.B.
        • Popescu N.C.
        • Weinberg R.A.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 12606-12611
        • Stewart S.A.
        • Ben-Porath I.
        • Carey V.J.
        • O'Connor B.F.
        • Hahn W.C.
        • Weinberg R.A.
        Nat. Genet. 2003; 33: 492-496
        • Stewart S.A.
        • Dykxhoorn D.M.
        • Palliser D.
        • Mizuno H.
        • Yu E.Y.
        • An D.S.
        • Sabatini D.M.
        • Chen I.S.
        • Hahn W.C.
        • Sharp P.A.
        • Weinberg R.A.
        • Novina C.D.
        RNA. 2003; 9: 493-501
        • Stucki M.
        • Jónsson Z.O.
        • Hübscher U.
        J. Biol. Chem. 2001; 276: 7843-7849
        • Brush G.S.
        • Kelly T.J.
        • Stillman B.
        Methods Enzymol. 1995; 262: 522-548
        • Kennedy B.K.
        • Barbie D.A.
        • Classon M.
        • Dyson N.
        • Harlow E.
        Genes Dev. 2000; 14: 2855-2868
        • Sengupta S.
        • Linke S.P.
        • Pedeux R.
        • Yang Q.
        • Farnsworth J.
        • Garfield S.H.
        • Valerie K.
        • Shay J.W.
        • Ellis N.A.
        • Wasylyk B.
        • Harris C.C.
        EMBO J. 2003; 22: 1210-1222
        • Sharma S.
        • Sommers J.A.
        • Gary R.K.
        • Friedrich-Heineken E.
        • Hübscher U.
        • Brosh Jr., R.M.
        Nucleic Acids Res. 2005; 33: 6769-6781
        • Shen B.
        • Nolan J.P.
        • Sklar L.A.
        • Park M.S.
        J. Biol. Chem. 1996; 271: 9173-9176
        • Stucki M.
        • Stagljar I.
        • Jónsson Z.O.
        • Hübscher U.
        Prog. Nucleic Acid Res. Mol. Biol. 2001; 65: 261-298
        • Zheng L.
        • Dai H.
        • Zhou M.
        • Li M.
        • Singh P.
        • Qiu J.
        • Tsark W.
        • Huang Q.
        • Kernstine K.
        • Zhang X.
        • Lin D.
        • Shen B.
        Nat. Med. 2007; 13: 812-819
        • Liu R.
        • Qiu J.
        • Finger L.D.
        • Zheng L.
        • Shen B.
        Nucleic Acids Res. 2006; 34: 1772-1784
        • Muftuoglu M.
        • Wong H.K.
        • Imam S.Z.
        • Wilson 3rd, D.M.
        • Bohr V.A.
        • Opresko P.L.
        Cancer Res. 2006; 66: 113-124
        • van Overbeek M.
        • de Lange T.
        Curr. Biol. 2006; 16: 1295-1302
        • Undarmaa B.
        • Kodama S.
        • Suzuki K.
        • Niwa O.
        • Watanabe M.
        Biochem. Biophys. Res. Commun. 2004; 315: 51-58
        • Durkin S.G.
        • Glover T.W.
        Annu. Rev. Genet. 2007; 41: 169-192
        • Branzei D.
        • Foiani M.
        Curr. Opin. Cell Biol. 2005; 17: 568-575
        • Xu L.
        • Blackburn E.H.
        Mol. Cell. 2007; 28: 315-327
        • Masutomi K.
        • Yu E.Y.
        • Khurts S.
        • Ben-Porath I.
        • Currier J.L.
        • Metz G.B.
        • Brooks M.W.
        • Kaneko S.
        • Murakami S.
        • DeCaprio J.A.
        • Weinberg R.A.
        • Stewart S.A.
        • Hahn W.C.
        Cell. 2003; 114: 241-253
        • Ivessa A.S.
        • Zhou J.Q.
        • Schulz V.P.
        • Monson E.K.
        • Zakian V.A.
        Genes Dev. 2002; 16: 1383-1396
        • Lillard-Wetherell K.
        • Machwe A.
        • Langland G.T.
        • Combs K.A.
        • Behbehani G.K.
        • Schonberg S.A.
        • German J.
        • Turchi J.J.
        • Orren D.K.
        • Groden J.
        Hum. Mol. Genet. 2004; 13: 1919-1932
        • Opresko P.L.
        • Otterlei M.
        • Graakjaer J.
        • Bruheim P.
        • Dawut L.
        • Kølvraa S.
        • May A.
        • Seidman M.M.
        • Bohr V.A.
        Mol. Cell. 2004; 14: 763-774
        • Poulet A.
        • Buisson R.
        • Faivre-Moskalenko C.
        • Koelblen M.
        • Amiard S.
        • Montel F.
        • Cuesta-Lopez S.
        • Bornet O.
        • Guerlesquin F.
        • Godet T.
        • Moukhtar J.
        • Argoul F.
        • Déclais A.C.
        • Lilley D.M.
        • Ip S.C.
        • West S.C.
        • Gilson E.
        • Giraud-Panis M.J.
        EMBO J. 2009; 28: 641-651
        • Dhillon K.K.
        • Sidorova J.
        • Saintigny Y.
        • Poot M.
        • Gollahon K.
        • Rabinovitch P.S.
        • Monnat Jr., R.J.
        Aging Cell. 2007; 6: 53-61
        • Sidorova J.M.
        • Li N.
        • Folch A.
        • Monnat Jr., R.J.
        Cell Cycle. 2008; 7: 796-807
        • Nikolova T.
        • Christmann M.
        • Kaina B.
        Anticancer Res. 2009; 29: 2453-2459
        • Moreau S.
        • Morgan E.A.
        • Symington L.S.
        Genetics. 2001; 159: 1423-1433
        • Kang H.Y.
        • Choi E.
        • Bae S.H.
        • Lee K.H.
        • Gim B.S.
        • Kim H.D.
        • Park C.
        • MacNeill S.A.
        • Seo Y.S.
        Genetics. 2000; 155: 1055-1067
        • Bae S.H.
        • Seo Y.S.
        J. Biol. Chem. 2000; 275: 38022-38031
        • Ayyagari R.
        • Gomes X.V.
        • Gordenin D.A.
        • Burgers P.M.
        J. Biol. Chem. 2003; 278: 1618-1625
        • Kao H.I.
        • Campbell J.L.
        • Bambara R.A.
        J. Biol. Chem. 2004; 279: 50840-50849
        • Dantzer F.
        • Giraud-Panis M.J.
        • Jaco I.
        • Amé J.C.
        • Schultz I.
        • Blasco M.
        • Koering C.E.
        • Gilson E.
        • Ménissier-de Murcia J.
        • de Murcia G.
        • Schreiber V.
        Mol. Cell. Biol. 2004; 24: 1595-1607
        • Ye J.Z.
        • de Lange T.
        Nat. Genet. 2004; 36: 618-623
        • Bryant H.E.
        • Petermann E.
        • Schultz N.
        • Jemth A.S.
        • Loseva O.
        • Issaeva N.
        • Johansson F.
        • Fernandez S.
        • McGlynn P.
        • Helleday T.
        EMBO J. 2009; 28: 2601-2615
        • Sun H.
        • Karow J.K.
        • Hickson I.D.
        • Maizels N.
        J. Biol. Chem. 1998; 273: 27587-27592
        • Davies S.L.
        • North P.S.
        • Hickson I.D.
        Nat. Struct. Mol. Biol. 2007; 14: 677-679