TRF2 overexpression diminishes repair of telomeric single-strand breaks and accelerates telomere shortening in human fibroblasts

Mechanisms of Ageing and Development 128 (2007) 340–345 www.elsevier.com/locate/mechagedev

TRF2 overexpression diminishes repair of telomeric single-strand breaks and accelerates telomere shortening in human fibroblasts Torsten Richter, Gabriele Saretzki, Glyn Nelson, Mathias Melcher, Sharon Olijslagers, Thomas von Zglinicki * Henry Wellcome Biogerontology Laboratory and Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, University of Newcastle upon Tyne, UK Received 27 June 2006; received in revised form 14 February 2007; accepted 28 February 2007 Available online 4 March 2007

Abstract Repair of single strand breaks in telomeric DNA is less efficient than in other genomic regions. This leads to an increased vulnerability of telomeric DNA towards damage induced by reactive oxygen species (ROS) and to accelerated telomere shortening under oxidative stress. The causes for the diminished repair efficacy in telomeres are unknown. We show here that overexpression of the telomere-binding protein TRF2 further reduces telomeric, but not genomic, single strand break repair. This suggests the possibility of strand break repair in telomeres being sterically hindered by the three-dimensional structure of the telomere DNA–protein complex and explains the effect of TRF2 on telomere shortening rates in telomerase-negative cells. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Senescence; Telomeres; TRF2; Reactive oxygen; Fibroblast; DNA repair; Single strand breaks

1. Introduction Cells lacking the enzyme telomerase shorten their telomeres during each round of DNA replication. This occurs partly because of the inability of DNA polymerases to replicate the lagging strand to the very end due to the so-called ‘‘endreplication problem’’ (Olovnikov, 1996). In addition, oxidative stress determines the rate of telomere shortening to a large extent. Earlier studies showed a positive correlation between cellular levels of oxidative stress and telomere shortening rates. For instance, telomere shortening in human MRC-5 fibroblasts was slower in cells cultured under normoxia (20% ambient oxygen partial pressure) than in those continuously grown under hyperoxia (40% oxygen). Telomere shortening could be slowed down further by treatment with antioxidants (Kashino et al., 2003; Kurz et al., 2004; Liu et al., 2002; Saretzki et al., 2003; Serra et al., 2003; von Zglinicki, 2002). DNA replication is a * Corresponding author at: Henry Wellcome Laboratory for Biogerontology Research, Institute for Ageing and Health, Newcastle, General Hospital, Newcastle upon Tyne NE4 6BE, UK. Tel.: +44 191 256 3310; fax: +44 191 256 3445. E-mail address: [email protected] (T. von Zglinicki). 0047-6374/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2007.02.003

prerequisite both for the end replication problem and for stressdependent telomere shortening. Growth-arrested fibroblasts accumulate DNA single-strand-breaks (SSB) preferentially in their telomeres under the influence of reactive oxygen species (ROS). Repair of SSBs is significantly less efficient in telomeres as compared to bulk DNA or to non-coding interstitial DNA. Significant amounts of telomeric SSBs are not repaired even days and weeks after induction in growth-arrested human fibroblasts (Petersen et al., 1998). However, if the cells are allowed to divide, SSBs are lost while telomere shortening is accelerated (Sitte et al., 1998). These data suggested that accumulation of oxidative lesions, and especially SSBs, might interfere with the full replication of telomeric DNA. It was suggested that SSBs might abort DNA replication in the close vicinity of the strand terminus, leaving one replicated strand truncated and the other with a SSB where the newly synthesized strand meets the non-replicated strand (Richter and von Zglinicki, 2006). However, so far the evidence for SSBs resulting in stalling or abort of DNA replication is sparse. It is also not clear why SSB are less efficiently repaired in telomeres as compared to non-telomeric genomic DNA. SSB can occur as direct consequence of oxidative attack, but are more frequently produced as intermediates in base excision

T. Richter et al. / Mechanisms of Ageing and Development 128 (2007) 340–345

repair (BER) of oxidatively modified bases or abasic sites (Sliwinski and Blasiak, 2005). About 80% of SSBs in the genome are repaired by an enzyme complex involving POLb, XRCC1 and Lig3 (Caldecott, 2001). Remaining SSBs are repaired in a replication-dependent manner by a complex encompassing the ring-shaped, trimeric DNA polymerase factor PCNA together with APE1, POLd/e, Fen1 and Lig1. This same complex can also be involved in homologous recombination to re-establish DNA replication when the replication-coupled repair failed. Telomeres are highly complex, spatially organized structures (de Lange, 2005). At least three different proteins, TRF1, TRF2 and Pot1 directly bind to TTAGGG repeats, while three additional telomeric proteins, TIN2, TPP1 and Rap1, bind to the above proteins and stabilize the telomeric complex. Facilitated by protein–DNA interactions, especially with TRF2, DNA is folded back in a d-loop/t-loop structure (Stansel et al., 2001). Finally, telomerase can bind to the single-stranded telomeric terminus and contribute to telomere capping (de Lange, 2005). Thus, it can be expected that expression of telomere-binding proteins might interfere with the efficacy of telomeric SSB repair. Stabilization of the telomere higher order structure might easily lead to a decreased accessibility of telomeric DNA to DNA repair complexes. In fact, over-expression of TRF2 has been shown not only to stabilize the telomeric loop and to postpone uncapping, but also to accelerate telomere shortening (Karlseder et al., 2002). We therefore examined the effect of TRF2 expression on telomeric SSB repair and found that the acceleration of telomere loss by TRF2 can be explained by the inhibitory effect of this protein on telomeric SSB repair. 2. Materials and methods 2.1. Cell culture, transduction and treatments Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), containing 10% fetal calf serum, 1% glutamine and 1% penicillin–streptomycin. Human MRC-5 fibroblasts were obtained from EACC. Retrovirus particles containing either pLCP-N-myc-TRF2 (kind gift from T. de Lange) or empty vector were produced in FNX producer cells. MRC-5 fibroblasts at a population doubling (PD) level of 28–35 were transduced with the supernatants and selected with puromycin for 10 days. Puromycin-resistant cells were pooled, and a starting PD = 1 was assigned to the cells when they formed the first confluent dish. Cells were treated with hydrogen peroxide (400 mM in serum-free medium) for 1 h and genomic and telomeric DNA strand breaks were measured before the treatment, immediately after it and after 24 h repair (see below).

2.2. FADU assay Single and double stand breaks in genomic DNA were measured using the semi-automated Fluorescence Detected Alkaline DNA Unwinding (FADU) assay as described (Brabeck et al., 2003). Briefly, cells were washed in PBS and lysed in 0.25 M meso-inositol, 1M MgCl2, 10 mM Na2PO4/NaH2PO4, pH 7.2. The lysed cells were transferred onto a 96-well plate (replicates of 8–10), kept at 4 8C, and denaturation buffer (9 M urea, 10 mM NaOH, 25 mM CDTA (trans-1,2-diaminocylohexan-N,N,N0 N0 -tetraacetic acid), 0,1% SDS) was added, followed by an alkaline solution (200 mM NaOH, 40% denaturation buffer) at 37 8C for 90 min. DNA was stained with the intercalating dye SYBR Green (diluted 1:250,000 in 13 mM NaOH) and fluorescence, originating from

341

double-stranded DNA, was measured in a fluorescence reader Spectrafluor Plus (Tecan, Crailsheim, Germany) at 492 nm exitation and 520 nm emission. The fluorescence intensity is a measure of the integrity of double-stranded DNA and is thus inversely correlated to the number of DNA stand breaks present at the time of lysis. Percentage of DNA repair was expressed as 100  (Px,t  Px,0)/ (P0  Px,0), with P0 being the fluorescence intensity of the untreated control sample, Px,0 the intensity in the treated sample immediately after treatment, and Px,t the fluorescence intensity of the treated sample after repair for the time t (24 h).

2.3. Western blotting SDS gel electrophoresis was performed in an 8% acryl amide gel with a protein content of 45 mg at 120 V (constant voltage) in 0.025 M Tris pH 8.3, 0.192 M glycine, 0.1% SDS. Gels were blotted on PVDF cellulose membrane (Amersham). The membrane was blocked (1:10 Rothiblock (Roth), 0.5% BSA for 1 h at room temperature), incubated with the primary antibody (1:1000 in blocking solution at 4 8C, overnight). A secondary horseradish peroxidase conjugated antibody (1:1000, 1 h at room temperature) was used and detected using the ECLplus Western Blotting Detection System (Amersham). The luminescence signal was quantified using a LAS-1000 (Fuji).

2.4. Immuno-FISH Immuno-FISH combines immunofluorescence with in situ hybridization of peptide nucleic acid (PNA) to telomeric DNA to show localization of a certain protein (here TRF2) with respect to telomeres. Immuno-FISH was performed on methanol acetic acid (3:1) fixed cells on cover-slips. For immunocytochemistry for TRF2, anti-TRF2 (mouse monoclonal IgG1, Upstate Cat # 05-521, dilution 1:1000) and FITC-conjugated anti-mouse IgG1 (1:2000) were each applied for 1 h at room temperature. After dehydration in ethanol series (70, 90 and 100%), in situ hybridization (FISH) was performed with 0.5 mg/ml of PNA probe (Applied Biosystem) for 3 min at 80 8C in 1 mM Tris pH 7.2, 2.14 mM magnesium chloride, 0.77 mM citric acid, 7.02 mM sodium hydrogen phosphate, 70% formamide and 5% Blocking Reagent (Roche), followed by 2 h in a humid chamber at room temperature. Coverslips were washed two times for 15 min in 70% formamide, 10 mM Tris pH 7.2, 0.1 % BSA, three times for 5 min in PBS, 0.05% Tween and dehydrated in ethanol series. To increase the signal, the secondary antibody incubation was repeated. DAPI staining solution (PARTEC; 05-5001) was applied for 10 min and the slides were washed three times with PBS for 5 min. Images were taken in confocal mode (Zeiss LSM 510 META).

2.5. Measurement of telomere restriction fragment length DNA for in-gel hybridization was prepared in 2% agarose plugs in 10 mM Tris pH 7.2, 20 mM NaCl, 50 mM EDTA using plug molds (Bio-Rad Laboratories) in a concentration of 1 million cells per plug. Plugs were digested with 250 ml proteinase K solution (20 mg/ml in 100 mM EDTA pH 8, 0.20% sodium deoxycholate, 1% sodium laurylsarcosine) for 48 h at 37 8C and washed in 20 mM Tris pH 8, 50 mM EDTA. After digestion overnight with 20 units of Hinf 1 in 100 ml buffer H (50 mM Tris–Cl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, pH 7.5), pulsed field gel electrophoresis was performed with quarter plugs in an 1% agarose gel in 0.5 TBE using Hind 1 digested phage lambda DNA as a marker. Electrophoresis conditions were initial switch: 2 s, final switch: 10 s, 3.5 V for 17 h at 14 8C. Gels were dried for 30 min at 37 8C, denatured for 30 min in 0.5 M NaOH, 1.5 M NaCl, neutralized for 30 min in 0.5 M Tris pH 7.4, 1.5 M NaCl and preincubated in rotating glass jars with 10 ml RapidHyb buffer (Amersham Bioscience) at 43 8C for 1 h. The gels were hybridized at 43 8C overnight with 10 ml probe oligonucleotide (100 pmol (CCCTAA)4 end-labeled with 100 mCi 32P in RapidHyb buffer). Washes were done in 2 SSC buffer (0.03 M Sodium Citrate pH 7.4, 0.3 M NaCl) at room temperature for 10 min and four times in 0.2 SSC buffer at 43 8C for 1 h. Signals were detected in a Storm scanner 820 (Molecular Dynamics). Fragment sizes were determined as size-weighted average in Advanced Image Data Analyser (AIDA, Raytest Isotopenmessgeraete GmbH) using 1D evaluation mode.

342

T. Richter et al. / Mechanisms of Ageing and Development 128 (2007) 340–345

2.6. S1 nuclease assay SSB in telomeric regions were determined as S1 nuclease-sensitive sites as described earlier (Petersen et al., 1998). Briefly, Hinf1 digested plugs were preincubated at 37 8C for 1 h in 300 ml S1 buffer (Amersham) containing 3.3 mM sodium acetate, 5 mM NaCl, 0.1 mM ZnCl2, pH 4.5. Afterwards plugs were quartered and incubated at 37 8C for 5 h with 0, 1, 2 and 5 U S1 nuclease (Amersham) per mg DNA in S1 buffer. The reaction was stopped using 25 mM EDTA. After fragment size determination the number of single strand breaks per Mbp DNA was calculated by using Eq. (1) L0  Lx nx ¼ L0 Lx

(1)

where L0 is the mean fragment length of DNA in Mbp at a S1 nuclease concentration of 0 U and Lx is the mean fragment length at a nuclease concentration of x U. The data were fitted to a hyperbolic curve according to Eq. (2) using the linearized Eq. (3) to determine the constants. The asymptotic maximum of this curve N was taken as the estimate of S1-sensitive sites. Nx x þ K0

(2)

1 K0 1 1 ¼  þ nx N x N

(3)

nx ¼

K0 is the equivalent to the Michaelis–Menten constant and represents the S1 nuclease concentration at which half of all S1-sensitive sites are converted into double strand breaks. N is the maximal number of S1-sensitive sites (SSB) per Mbp. Percentage of telomeric DNA repair was calculated as described for the FADU assay.

2.7. Senescence-associated b-galactosidase staining Cell were fixed in 1% formaldehyde/PBS for 5 min at room temperature, washed twice in PBS and stained overnight at 37 8C in 8.8 ml b-galactosidase

buffer (150 mM sodium chloride, 2 mM magnesium chloride, 40 mM citric acid, 12 mM sodium phosphate pH 6.0), 1 ml potassium ferro-cyanide preparation (2.11 g potassium hexacyanoferrat(II)trihydrate, 1.65 g potassium hexacyanoferrat(III)trihydrate, 100 ml water) and 200 ml X-gal (20 mg/ml in dimethylformamide).

3. Results Overexpression of TRF2 in human IMR90 and BJ fibroblasts accelerated telomere shortening by 50–80% and caused shorter telomeres at senescence, the net result being a slight increase of replicative lifespan. This was in line with the idea that TRF2 stabilizes the telomeric cap and thus delays replicative senescence (Karlseder et al., 2002). To test the generality of these findings, we infected human embryonic lung MRC5 fibroblasts with a retrovirus carrying a TRF2 expression vector. This increased TRF2 protein levels by about 100% above vector-only transfected controls (MRC-v, Fig. 1A). TRF2 antibody staining resulted in a punctuate nuclear pattern with about half of the TRF2 foci co-localising with telomeres in MRC-TRF2 (Fig. 1B). This was not different from the localization seen in wild type cells (not shown). It has been shown recently that phosphorylated TRF2 can accumulate at non-telomeric DNA damage sites (Tanaka et al., 2005), which might explain the non-telomeric TRF2 foci observed. At 6 PD after selection, MRC-v cells showed senescent morphology characterized by enlarged, flattened cells with irregular shape, while MRC-TRF2 cells maintained growth and corresponding morphology for more than 7 PD following selection (Fig. 1C).

Fig. 1. Overexpressed TRF2 co-localizes with telomeres and delays senescence of MRC5 human fibroblasts. (A) Western blots of 45 mg total protein from MRCTRF2 and MRC-v fibroblasts were probed with anti-TRF2 (top) and anti-cdk4 (bottom, loading control). (B) Immuno-FISH with a PNA telomeric probe (red) and anti-TRF2 (green) showed frequent telomeric location of TRF2 (see higher magnification images of boxed areas 1 and 2) in MRC-TRF2. (C) Morphology (left) and sen-b gal activity (right) of MRC-TRF2 fibroblasts at 7 PD after selection (top) and MRC-v cells at 6 PD after selection (bottom). The bar graph shows the frequency of sen-b-gal positive cells. Data are mean  S.D. from five images each. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

T. Richter et al. / Mechanisms of Ageing and Development 128 (2007) 340–345

343

Fig. 2. TRF2 overexpression accelerates telomere shortening. (A) Representative telomere in-gel hybridization images from MRC-TRF2 and MRC-v fibroblasts. DNA was isolated at the PDs after transfection as indicated on top of the lanes. (B) Telomere shortening in MRC-TRF2 (top) and MRC5-v cells (bottom). Data are mean  S.E.M. from triplicate experiments. Linear regression and 95% confidence intervals are shown. Telomere shortening rate (slope of the regression line) and Spearman’s regression coefficient are indicated.

At the same time points, the frequency of cells positive for senescence-associated b-galactosidase, a marker for cellular senescence, was significantly lower in MRC-TRF2 cells as compared to MRC-v fibroblasts (Fig. 1C). Moreover, MRCTRF2 cells reached a higher cumulative PD than MRC-v cells (supplementary material, Fig. S1). Thus, TRF2 overexpression delayed senescence also in MRC5 fibroblasts. Telomeres in MRC-TRF2 cells shortened faster than in controls (Fig. 2). Quantitative evaluation of triplicate Southern blots resulted in telomere shortening rates of 96 bp/PD for MRC-TRF2 fibroblasts, while the rate for MRC-v cells was 76 bp/PD in accordance with published values for wild-type MRC5 cells (Keys et al., 2004). Both regressions are significantly different ( p < 0.001, multiple ANOVA), largely due to a higher degree of telomere shortening during selection and until a sufficient number of cells for analysis was obtained (Fig. 2). DNA strand break induction and repair were measured in confluent cells at 3 PD after selection. At this time point, both strains proliferated at about the same rate. We first measured strand break induction and repair in the whole of the genome using an automated FADU (fluorescent alkaline DNA unwinding) assay. There was no significant difference between MRC-TRF2 and MRC-v cells in the frequency of induced strand breaks (Fig. 3A,1). Repair activities were also equal in MRC-v and MRC-TRF2 (Fig. 3A,2). Moreover, induction of

bulk DNA damage by 4Gy ionizing radiation and subsequent repair activity did not differ between MRC-TRF2 and MRC-v cells (data not shown). We conclude that overexpression of TRF2 did not significantly modify SSB repair in the bulk of the genomic DNA. We next measured the oxidative induction of SSBs and their repair specifically in telomeres. We had shown before that oxidatively induced SSBs were completely repaired after 24 h in interstitial non-transcribed DNA, but not in telomeres (Petersen et al., 1998). Thus, we isolated DNA before and after hydrogen peroxide treatment and 24 h later and determined frequencies of SSBs per unit of telomere length as described in Materials and Methods. Vector-only controls and TRF2 overexpressing cells showed equal basal levels of S1-sensitive sites. Telomeres in TRF2 overexpressing cells were significantly less sensitive to oxidative damage than vector transfected controls (Fig. 3B,1). This might be due to a scavenging effect of abundant TRF2 proteins surrounding the telomeres. However, despite lower levels of induced telomeric damage in MRCTRF2 cells, damage was even less well repaired (Fig. 3B,1). While in vector-only controls nearly 50% of the induced damage is repaired after 24 h, the repair efficiency decreased to less than 20% in TRF2 overexpressing cells (Fig. 3B,2). Thus, we conclude that overexpression of wild-type TRF2 results in compromised SSB repair specifically in telomeres. It should be stressed that a direct quantitative comparison of repair

344

T. Richter et al. / Mechanisms of Ageing and Development 128 (2007) 340–345

Fig. 3. Overexpression of TRF2 compromises repair of telomeric, but not genomic SSBs. (A) (1) Genomic DNA damage and repair as measured by FADU assay in MRC5 vector controls (MRV-v) and MRC5 cells overexpressing TRF2 (MRC-TRF2). DNA integrity after alkaline unwinding is measured by the fluorescence intensity of the intercalating dye SYBR green. Black bars: untreated (control), open bars: treated with 400 mM H2O2 (damage), grey bars: 24 h repair (repair). Data are mean  S.E.M. from one representative experiment (eight parallel measurements). (2) Average percentage of DNA repair in genomic DNA calculated from three independent experiments. Data are mean  S.E.M. n.s, non-significant. (B) (1) Frequency of telomeric SSB (max. SSB per Mbp) measured by S1-nuclease assay in untreated cells (control), after treatment with 400 mM H2O2 (damage) and after 24 h repair in MRC-v and MRC-TRF2 cells. Data are mean  S.E.M. from one representative experiment (triplicate measurements). (2) Percentage of DNA repair in telomeric DNA calculated from three independent experiments. Data are mean  S.E.M. The star denotes p < 0.005.

efficiencies between bulk DNA and telomeres is not possible due to the different techniques employed. For instance, linearity of the FADU technique has not been established. Inhibition of TRF2, for instance by overexpression of a dominant-negative mutant, induces immediate telomeredependent senescence (van Steensel et al., 1998). Induction of senescence compromised DNA strand break repair all over the genome to a degree that prevented the assessment of a specific telomeric effect of TRF2 inhibition (data not shown). 4. Discussion It is well established that cellular TRF2 expression levels determine telomeric DNA damage signalling and induction of telomere-dependent senescence: Disruption of telomeric TRF2 binding by overexpression of a dominant-negative TRF2 mutant induces telomere-dependent DNA damage signalling and senescence even in cells with long telomeres (Takai et al., 2003), while overexpression of wild-type TRF2 allows further telomere shortening before senescence signalling is induced (Karlseder et al., 2002). This telomere capping effect of TRF2 has been suggested to be due to the stabilizing effect of TRF2 on the telomeric loop structure (Griffith et al., 1999). It has also been shown that higher levels of TRF2 result in faster shortening of telomeres (Karlseder et al., 2002), and this was confirmed in the present study. Our data now show that

expression of TRF2 influences the efficacy of telomeric SSB repair in human fibroblasts. Higher levels of TRF2, which stabilize the telomere cap, result in less efficient repair of oxidatively induced telomeric single strand breaks, but do not impact on DNA strand break repair in the bulk of the genome. It has been shown that both nucleotide excision repair (NER), which removes UV-induced lesions from DNA (Kruk et al., 1995), and late steps in base excision repair (BER), which are responsible for the induction and repair of SSB after oxidative base modification (Opresko et al., 2005; Petersen et al., 1998; Sitte et al., 1998), are less efficient in telomeres than in the bulk of the genome. Recently, it was demonstrated that TRF2 overexpression accelerated telomere shortening in mice keratinocytes in a manner that is independent of telomerase but dependent on UV-induced DNA damage and its repair by NER. In this model, knock-down of XPF compensated for the TRF2-induced accelerated telomere shortening (Munoz et al., 2005). The ERCC1-XPF complex is the nuclease that incises the damaged DNA strand 50 from the DNA adduct, which together with the 30 specific nuclease XPG, forms a singlestranded gap as a repair intermediate. This gap is repaired by a DNA synthesis complex containing replication factor C, PCNA and DNA polymerase d or e (de Laat et al., 1999). Repair of SSB occurring as result of direct attack of free radicals to the sugar-phosphate backbone of the DNA or as BER intermediates is performed in a replication-coupled manner by a very similar

T. Richter et al. / Mechanisms of Ageing and Development 128 (2007) 340–345

complex containing PCNA and polymerase d/e or independent of replication by polymerase b in complex with XRCC1. Thus, it could be possible that binding of TRF2 to telomeres sterically hinders access of DNA repair complexes to telomeric SSB, which might be generated by different types of lesions either directly or as repair intermediates. The sliding clamp-type PCNA complex might be a prime candidate for sterical inhibition, and in fact, it has been shown that binding of TRF2 to telomeric repeats stalls the progress of the PCNA complex at the replication fork (Ohki and Ishikawa, 2004). A second possible mechanism by which TRF2 could impair telomeric repair is through its influence on the ATM kinase, which recognizes DNA damage. TRF2 was found to interact in vitro directly with ATM, inhibit its autophosphorylation and diminishes its general response to damaged DNA (Karlseder et al., 2004). However, while ATM does play a major role for DSB recognition, its involvement in repair of single strand DNA damage, which is the major type of damage considered here, has not been established. TRF2 also interacts with polymerase b (Fotiadou et al., 2004) and might thus impinge negatively on replicationindependent SSB repair. Interestingly, the presence of oxidative damage, for instance an 8-oxoGua modification or an abasic site, in DNA diminishes the binding of TRF2 to its telomeric substrate in vitro (Opresko et al., 2005). It is tempting to speculate that this might help at least some telomeric repair to happen. However this might be, the fact that TRF2 overexpression specifically diminishes SSB repair in telomeres, together with earlier data showing that SSBs in telomeres are transformed into telomere shortening during DNA replication (Sitte et al., 1998), appears sufficient to explain the effect of TRF2 on telomere shortening rates in primary, telomerasenegative human cells. Acknowledgments The study was supported by programme grant 252 from Research into Ageing, UK. T.R. was supported by a grant from the Roland Ernst Stiftung fuer Gesundheitswesen, Germany. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mad.2007.02.003. References Brabeck, C., Pfeiffer, R., Leake, A., Beneke, S., Meyer, R., Burkle, A., 2003. LSelegiline potentiates the cellular poly(ADP-ribosyl)ation response to ionizing radiation. J. Pharmacol. Exp. Ther. 306, 973–979. Caldecott, K.W., 2001. Mammalian DNA single-strand break repair: an Xra(y)ted affair. Bioessays 23, 447–455. de Laat, W.L., Jaspers, N.G.J., Hoeijmakers, J.H.J., 1999. Molecular mechanism of nucleotide excision repair. Genes Dev. 13, 768–785. de Lange, T., 2005. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 19, 2100–2110.

345

Fotiadou, P., Henegariu, O., Sweasy, J.B., 2004. DNA polymerase beta interacts with TRF2 and induces telomere dysfunction in a murine mammary cell line. Cancer Res. 64, 3830–3837. Griffith, J.D., Comeau, L., Rosenfield, S., Stansel, R.M., Bianchi, A., Moss, H., de Lange, T., 1999. Mammalian telomeres end in a large duplex loop. Cell 97, 503–514. Karlseder, J., Hoke, K., Mirzoeva, O.K., Bakkenist, C., Kastan, M.B., Petrini, J.H., de Lange, T., 2004. The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response. PLoS Biol. 2, E240. Karlseder, J., Smogorzewska, A., de Lange, T., 2002. Senescence induced by altered telomere state, not telomere loss. Science 295, 2446–2449. Kashino, G., Kodama, S., Nakayama, Y., Suzuki, K., Fukase, K., Goto, M., Watanabe, M., 2003. Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells. Free Radic. Biol. Med. 35, 438–443. Keys, B., Serra, V., Saretzki, G., Von Zglinicki, T., 2004. Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-3’-overhang. Aging Cell 3, 103–109. Kruk, P.A., Rampino, N.J., Bohr, V.A., 1995. DNA damage and repair in telomeres: relation to aging. Proc. Natl. Acad. Sci. U.S.A. 92, 258–262. Kurz, D.J., Decary, S., Hong, Y., Trivier, E., Akhmedov, A., Erusalimsky, J.D., 2004. Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells. J. Cell Sci. 117, 2417–2426. Liu, L., Trimarchi, J.R., Smith, P.J., Keefe, D.L., 2002. Mitochondrial dysfunction leads to telomere attrition and genomic instability. Aging Cell 1, 40–46. Munoz, P., Blanco, R., Flores, J.M., Blasco, M.A., 2005. XPF nucleasedependent telomere loss and increased DNA damage in mice overexpressing TRF2 result in premature aging and cancer. Nat. Genet. 37, 1063–1071. Ohki, R., Ishikawa, F., 2004. Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats. Nucleic Acids Res. 32, 1627–1637. Olovnikov, A.M., 1996. Telomeres, telomerase, and aging: origin of the theory. Exp. Gerontol. 31, 443–448. Opresko, P.L., Fan, J., Danzy, S., Wilson 3rd, D.M., Bohr, V.A., 2005. Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2. Nucleic Acids Res. 33, 1230–1239. Petersen, S., Saretzki, G., von Zglinicki, T., 1998. Preferential accumulation of single-stranded regions in telomeres of human fibroblasts. Exp. Cell Res. 239, 152–160. Richter, T., von Zglinicki, T., 2006. Oxidative DNA damage and telomere shortening. In: Mark Evans, M.C. (Ed.), Oxidative Damage to Nucleic Acids. Landes Bioscience, Georgetown, TX, pp. 1–10. Saretzki, G., Murphy, M.P., von Zglinicki, T., 2003. MitoQ counteracts telomere shortening and elongates lifespan of fibroblasts under mild oxidative stress. Aging Cell 2, 141–143. Serra, V., von Zglinicki, T., Lorenz, M., Saretzki, G., 2003. Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening. J. Biol. Chem. 278, 6824–6830. Sitte, N., Saretzki, G., von Zglinicki, T., 1998. Accelerated telomere shortening in fibroblasts after extended periods of confluency. Free Radic. Biol. Med. 24, 885–893. Sliwinski, T., Blasiak, J., 2005. Base excision repair. Postepy Biochem. 51, 120–129. Stansel, R.M., de Lange, T., Griffith, J.D., 2001. T-loop assembly in vitro involves binding of TRF2 near the 30 telomeric overhang. EMBO J. 20, 5532–5540. Takai, H., Smogorzewska, A., de Lange, T., 2003. DNA damage foci at dysfunctional telomeres. Curr. Biol. 13, 1549–1556. Tanaka, H., Mendonca, M.S., Bradshaw, P.S., Hoelz, D.J., Malkas, L.H., Meyn, M.S., Gilley, D., 2005. DNA damage-induced phosphorylation of the human telomere-associated protein TRF2. Proc. Natl. Acad. Sci. U.S.A. 102, 15539–15544. van Steensel, B., Smogorzewska, A., de Lange, T., 1998. TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413. von Zglinicki, T., 2002. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27, 339–344.