The Werner Syndrome Helicase and Exonuclease Cooperate to Resolve Telomeric D Loops in a Manner Regulated by TRF1 and TRF2

Molecular Cell, Vol. 14, 763–774, June 18, 2004, Copyright 2004 by Cell Press

The Werner Syndrome Helicase and Exonuclease Cooperate to Resolve Telomeric D Loops in a Manner Regulated by TRF1 and TRF2 Patricia L. Opresko,1 Marit Otterlei,2 Jesper Graakjær,3 Per Bruheim,2 Lale Dawut,1 Steen Kølvraa,3 Alfred May,1 Michael M. Seidman,1 and Vilhem A. Bohr1,* 1 Laboratory of Molecular Gerontology National Institute on Aging National Institutes of Health Baltimore, Maryland 21224 2 Department of Cancer Research and Molecular Medicine Norwegian University of Science and Technology N-7489 Trondheim Norway 3 Institute of Human Genetics University of Aarhus 8000 Aarhus C Denmark

Summary Werner syndrome (WS) is characterized by features of premature aging and is caused by loss of the RecQ helicase protein WRN. WS fibroblasts display defects associated with telomere dysfunction, including accelerated telomere erosion and premature senescence. In yeast, RecQ helicases act in an alternative pathway for telomere lengthening (ALT) via homologous recombination. We found that WRN associates with telomeres when dissociation of telomeric D loops is likely during replication and recombination. In human ALT cells, WRN associates directly with telomeric DNA. The majority of TRF1/PCNA colocalizing foci contained WRN in live S phase ALT cells but not in telomerase-positive HeLa cells. Biochemically, the WRN helicase and 3ⴕ to 5ⴕ exonuclease act simultaneously and cooperate to release the 3ⴕ invading tail from a telomeric D loop in vitro. The telomere binding proteins TRF1 and TRF2 limit digestion by WRN. We propose roles for WRN in dissociating telomeric structures in telomerase-deficient cells. Introduction Werner syndrome (WS) is a genomic instability disorder resulting from defects in the WRN gene, a RecQ DNA helicase (Yu et al., 1996). Deficiencies in human RecQ helicases BLM and RecQL4 are responsible for Bloom and Rothmund-Thomson syndromes, respectively; all three disorders show increased cancer (reviewed in Hickson, 2003). WS patients display the early onset of many age-associated pathologies, including greying and loss of hair, wrinkling of the skin, cataracts, type II diabetes, osteoporosis, and cardiovascular disease (Martin, 1978). WS cells are marked by increased chromosomal rearrangements and deletions, an extended S phase, premature senescence, and defects in homolo*Correspondence: [email protected]

gous recombination (HR) (reviewed in Opresko et al., 2003). However, the molecular basis of the genomic instability and premature senescence in cultured WS cells is still not clear. The WRN gene encodes a 3⬘ to 5⬘ DNA helicase that unwinds a variety of DNA substrates, including Holliday junctions, and non-B form DNA, such as G quadruplexes (Mohaghegh et al., 2001). The WRN protein is unique among human RecQ helicases in that it also contains a 3⬘ to 5⬘ exonuclease (Huang et al., 1998). Whether the WRN helicase and exonuclease cooperate to resolve DNA structures is unclear. WRN and BLM interact with the critical telomere repeat binding factor TRF2 (Opresko et al., 2002; Stavropoulos et al., 2002), suggesting a role for these helicases in telomere metabolism. The premature senescence observed in WS cells may be related to defects in telomere processing. Telomeres protect the ends of linear chromosomes and are important determinants of cellular life span. They end in a 3⬘ single strand G-rich tail that is proposed to invade duplex telomeric DNA resulting in a large t loop that is stabilized by an intratelomeric D loop (Griffith et al., 1999). Stabilization of the telomeric structure involves a complex of proteins, including TRF1 and TRF2. Telomere dysfunction results from progressive telomere erosion during cell division that occurs in normal somatic cells or from defects in telomere maintenance proteins and structure (De Lange, 2002). The expression of a TRF2 dominant-negative (DN) mutant, which fails to bind DNA, induces loss of the 3⬘ telomere tail, telomere end fusions, and apoptosis or senescence (Karlseder et al., 1999, 2002). WS fibroblasts display accelerated telomere erosion (Schulz et al., 1996), and WS lymphoblasts show erratic telomere length dynamics (Tahara et al., 1997). The forced expression of exogenous telomerase can bypass telomere-associated replicative senescence (Bodnar et al., 1998) and prevents premature senescence in WS fibroblasts (Wyllie et al., 2000). Premature senescent WS cells show features of telomere-induced replicative senescent cells (Davis et al., 2003). Collectively, these results suggest that senescence in WS cells is related to telomere defects. RecQ helicases may act in recombination at telomeres. A Rad50- and Rad52-dependent pathway for telomere lengthening occurs in telomerase-negative S. cerevisiae strains (Henson et al., 2002). The S. cerevisiae RecQ homolog Sgs1p is required for this pathway, and its role can be partially substituted by WRN (Johnson et al., 2001; Cohen and Sinclair, 2001; Huang et al., 2001). An analogous telomerase-independent pathway, alternative lengthening of telomeres (ALT), has been detected in immortalized human cell lines that lack telomerase activity (Yeager et al., 1999). These cells display unique nuclear foci termed ALT-associated PML bodies (AA-PML) that contain RAD52, RAD51, RPA, TRF1, TRF2, telomeric DNA (Yeager et al., 1999), and NBS1 and are enriched during the late S and G2 phases of the cell cycle (Wu et al., 2000). BLM and WRN also localize to these bodies (Yankiwski et al., 2000; Johnson

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et al., 2001) and interact with many of the components. The molecular basis of ALT is poorly understood but involves copying of telomeric DNA between telomeres (Dunham et al., 2000). These findings are consistent with models in which the 3⬘ telomeric tail invades the telomeric duplex of another chromosome, forming an intertelomeric D loop for use as a template. Intratelomeric D loops may also be important (Henson et al., 2002). Based on the above studies, we hypothesized that WRN may function at telomeres to dissociate telomeric end structures either for progression of the DNA replication fork and/or for recombination repair. Consistent with this, we find that WRN localizes to telomeric DNA in S phase ALT cells when resolution of telomeric D loops is likely required. We demonstrate that the WRN helicase and exonuclease cooperate to resolve a model telomeric D loop in vitro and are regulated by TRF1 and TRF2. Results WRN Localization in Live Cells Resolution of telomeric end structures during S phase is necessary for progression of the replication fork, and resolution of recombination intermediates likely occurs in ALT. Therefore, we examined WRN localization in S phase telomerase-negative U-2 OS cells (ALT) and telomerase-positive HeLa cells. Since WRN localizes to replication foci after damage (Constantinou et al., 2000) (Baynton et al., 2003), we wanted to minimize potential cellular damage. Construction of a U-2 OS cell line that stably expresses EYFP-WRN enabled us to view live cells, thereby avoiding any fixation artifacts. EYFP-WRN was primarily observed in nucleoli and also in multiple nuclear foci in about 10%–20% of the cells (Figure 1A), as reported for endogenous WRN in ALT cells (Johnson et al., 2001). To identify live S phase cells and to avoid potential DNA damage from synchronization treatments, we used HcRed proliferating cellular nuclear antigen (PCNA). Previous studies found that EGFP-PCNA forms distinct foci in S phase cells that colocalize completely with endogenous PCNA and bromo-deoxyuridine (BrdU) incorporation and, thus, represent sites of DNA replication (Leonhardt et al., 2000). In contrast, EGFP-PCNA showed a diffuse nuclear pattern in non-S phase cells. In control experiments, we determined that HcRed-PCNA localized in S and non-S phase U-2 OS cells similar to endogenous PCNA (Figure 1B). We found that fluorescent-tagged TRF1 was a good marker for telomeric DNA in live cells. EYFP-TRF1 stably expressed in U-2 OS cells formed foci that completely colocalized with endogenous TRF2 (Figure 1C, yellow foci). Metaphase spreads of these cells show that fluorescent-tagged TRF1 localized directly to the chromosomal ends (Figure 1D). EYFP-TRF1 colocalized completely with a CY3-telomeric probe, and we observed an excellent correlation (0.88) between the signal intensities for EYFP-TRF1 and the colocalizing CY3-telomeric probe (Figure 1E). WRN Colocalizes with TRF1 at PCNA Foci in S Phase ALT Cells To examine WRN localization in S phase ALT cells, we cotransfected U-2 OS cells expressing EYFP-WRN with

expression vectors for HcRed-PCNA and ECFP-TRF1. The fluorescent-tagged proteins were visualized in live, cycling cells by confocal microscopy in three separate channels. In representative S phase U-2 OS cells, EYFPWRN localized in the nucleoli, with a mean number of 16 ⫾ 5 (10–24, n ⫽ 10) foci. ECFP-TRF1 localized to a mean number of 27 ⫾ 5 (21–35) foci, and some cells displayed ECFP-TRF1 localization in nucleoli (Figure 2C), which may store excess TRF1. Pairwise comparisons of fluorescent-tagged proteins for each cell were conducted (Figure 2A) by assigning colors to each protein (green or red) to visualize colocalizing foci (shades of yellow). A significant fraction of WRN foci (mean, 70%) colocalized with TRF1, and a smaller fraction colocalized with PCNA (mean, 43%). Sites of triple staining were identified by PCNA and TRF1 colocalization (Figure 2Ai) and then asking if WRN was also present (Figure 2Af). The majority of TRF1/PCNA foci also contained WRN (mean, 81%) (Table 1), suggesting that WRN was likely to be present at replication sites in telomeres. Other functions are also likely, since only an average of 55% of the WRN/TRF1 foci colocalized with PCNA, although this number varied (27%–86%) (Table 1). Some colocalization of WRN and TRF1 also occurred in non-S phase ALT cells (Figure 2D). WRN Associates with Telomeric DNA in ALT Cells Although colocalization of WRN with TRF1 and TRF2 suggested a telomeric location, it was possible that the association could be outside of telomeric DNA. We had observed that the majority of the EYFP-WRN foci colocalized with an antibody against PML (60%) (Supplemental Figure S1 at http://www.molecule.org/cgi/ content/full/14/6/763/DC1), confirming localization in AA-PML bodies. We then stained cells expressing EYFP-WRN with a CY3-telomeric probe. Out of 245 cells that were positive for EYFP-WRN foci colocalizing with PML, we found that 114 (46%) displayed discernable colocalization (Figure 3Ac, yellow foci) between EYFPWRN (Figure 3Aa) and the CY3-telomere probe (Figure 3Ab). Within these cells, the percent of WRN foci colocalizing with telomeric DNA averaged 45% and varied from 20% to 100%, as this population included both S phase and non-S phase cells. WRN colocalized primarily with two classes of telomeric DNA: (1) those of average signal intensity (average length) and (2) those of high intensity (Figure 3B). The majority of the telomeres in the second class colocalized with WRN and AA-PML bodies. These may be very long telomeres, a cluster of telomeres perhaps engaged in recombination, or extrachromosomal telomeric DNA (Henson et al., 2002). To confirm WRN association with telomeric DNA in the ALT cells, we performed parallel chromatin immunoprecipitation (ChIP) on cells expressing either EYFP-WRN or EYFP-TRF1. Crosslinked protein-DNA complexes were immunoprecipitated and the DNA was analyzed by slot blot and quantitative hybridization with a telomeric probe and a probe against Alu repeat DNA as a negative control. Approximately 10% of the EYFPWRN and 4% of the EYFP-TRF1 protein were immunoprecipitated by an antibody against the EYFP tag (Figure 3C). The yield of precipitated telomeric DNA was approximately 3.8% with EYFP-TRF1 and 2.4% with EYFPWRN (lane 1) of total telomeric DNA (lane 3), a 6-fold

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Figure 1. Proper Localization of Fluorescent-Tagged Proteins in Live Cycling U-2 OS Cells (A) Confocal images of the most representative U-2 OS cells stably expressing EYFP-WRN (yellow) (Aa), whereas 10%–20% of the cells showed WRN in multiple foci (Ab). (B) Endogenous PCNA was detected by immunofluorescence in fixed cells (Ba and Bb). HcRed-PCNA was transiently expressed in live cycling cells (Bc and Bd). Confocal images of S phase cells (Ba and Bc) and non-S phase cells (Bb and Bd) are shown. (C) Colocalization of EYFP-TRF1 (Cb, shown as green) with endogenous immunostained CY3-TRF2 (Ca, red) is shown in the merged confocal image (Cc, yellow). (D) Metaphase spreads of U-2 OS cells stably expressing EYFP-TRF1 show fluorescent-tagged TRF1 (dark foci) at telomeric ends; the DNA was stained with dapi. (E) The signal intensity from the telomere probe was plotted against the signal intensity of colocalizing EYFP-TRF1 foci and the correlation coefficient was calculated (see Experimental Procedures).

increase above background (lane 2) (Figure 3D). After adjustment for the efficiency of protein immunoprecipitation (Figure 3C), we found that EYFP-TRF1 associated with a larger fraction of total telomeric DNA (near 76%), compared to EYFP-WRN (24%), as expected. In contrast, the protein-DNA complexes contained less than 0.1% of the total Alu repeat DNA. Thus, the enrichment for telomeric DNA in the WRN and TRF1 protein-DNA complexes was specific. WRN Localization in S Phase HeLa Cells In telomerase-positive S phase HeLa cells, transiently expressed EYFP-WRN localized primarily to the nucleoli (Supplemental Figures S2a–S2c on Molecular Cell’s website). Analyses of more than 100 HeLa cells cotransfected with EYFP-WRN and ECFP-PCNA showed that approximately 50% displayed no colocalization and the

other 50% showed very few (one to three) WRN and PCNA colocalizing foci. These foci likely represented sites of stalled replication forks due to endogenous DNA damage. After coexpression of ECFP-TRF1 and EYFPWRN in HeLa cells, some EYFP-WRN foci were in close proximity to ECFP-TRF1 foci (Supplemental Figure S2d– S2f on Molecular Cell’s website), but colocalization was rare in these cells. Thus, WRN was less likely to be present at telomeres during DNA replication in cells proficient for telomerase activity, at least in the absence of exogenous DNA damage. WRN Resolves a Telomeric D Loop Structure Our results suggest that WRN associates with telomeres when dissociation of telomeric D loops is likely required, either for replication and/or recombination. Thus, we investigated the biochemical action of WRN on model

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Figure 2. WRN Colocalizes with TRF1 at PCNA Foci in S Phase ALT Cells (A) Confocal images of representative S phase ALT cells. U-2 OS cells stably expressing EYFP-WRN (Aa and Ad) were cotransfected with expression vectors for ECFP-TRF1 (Ae and Ah) and HcRed-PCNA (Ab and Ag). Colors were assigned (green and red) to allow for identification of colocalizing foci (yellow, indicated by white arrows) (Ac, Af, and Ai) in pairwise comparisons. In a merge of all three (Aj), EYFP-WRN (green), ECFP-TRF1 (red), and HcRed-PCNA (purple), sites of triple colocalization appears white (arrows). (B and C) Shown are other examples of triple-stained S phase U-2 OS cells as in (A), with EYFP-WRN (green), ECFP-TRF1 (red), and HcRedPCNA (purple). White arrows indicate triple colocalization; green arrows indicate WRN and PCNA colocalization. (D) Shown is a confocal image of a triple-stained non-S phase U-2 OS cell, EYFP-WRN (green), ECFP-TRF1 (red), and HcRed-PCNA (purple). The arrows indicate sites of WRN and TRF1 colocalization.

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Table 1. Percent of WRN and TRF1 Foci Colocalization Alone and with PCNA

WRN TRF1 WRN/TRF1 WRN/PCNA TRF1/PCNA

WRN

TRF1

PCNA

– 42% (19–54) – – 81% (67–100)

70% (50–92) – – 82% (67–100) –

43% (28–58) 26% (15–42) 55% (27–86) – –

Values represent the mean percent and range (parentheses) for ten cells.

telomeric end structures in vitro. Previously, the WRN helicase and exonuclease were found to act independently on a short 22 bp D loop in vitro (Orren et al., 2002). We wanted to test the potential contribution of both WRN activities, simultaneously, on a model telomeric D loop. We designed a D loop consisting of a bubble with two 33 bp duplex arms and a 33 bp melted region in which an invading strand (INV) that mimics the

3⬘ telomeric tail was hybridized. The INV strand contained a few base pairs of unique sequence at the 3⬘ end to aid in proper alignment (Figure 4A). Analysis by native gel (Figure 4B) indicated that a D loop with a 5⬘ end labeled INV (lane 3) or bottom (BB) (lane 6) strand yielded a single species. Proper alignment of the telomeric repeats was confirmed with restriction enzymes (Figure 4C). WRN reactions were run on a native gel to display helicase activity (Figure 4D) and on a denaturing gel to visualize the 3⬘ to 5⬘ exonuclease products (Figure 4E). The substrate was not sufficiently unwound to release the full-length 67-mer INV strand with up to 6 nM WRN (Figure 4D). However, incubation with increasing amounts of WRN resulted in disappearance of the D loop substrate and the appearance of bands migrating below the fulllength INV strand. The WRN exonuclease initiated digestion at the INV 3⬘ end, and the helicase released the shortened INV strands, which were not digested further since the WRN exonuclease is largely inactive on ssDNA (Opresko et al., 2001). The most prominent products

Figure 3. WRN Associates with Telomeric DNA in ALT Cells (A) EYFP-WRN expressing U-2 OS cells (Aa) were stained with a CY3-PNA telomere probe (Ab). Sites of colocalization are indicated in the merge ([Ac], yellow foci). (B) In cells positive for WRN colocalization with PML and telomeric DNA (n ⫽ 114), the telomere probe signal intensity was quantitated and standardized for WRN colocalizing (n ⫽ 222) and noncolocalizing (n ⫽ 1769) telomeric foci (see Experimental Procedures); mean telomere length ⫽ 0. The percent of EYFP-WRN colocalizing (black bars) and noncolocalizing (white bars) telomere foci at each telomere signal intensity value was calculated as a function of total telomere foci (n ⫽ 1991). (C) ChIP assay protein recovery. Immunoprecipitated protein-DNA complexes from EYFP-WRN (top) or EYFP-TRF1 (bottom) expressing U-2 OS cells were probed by W. blot with an antibody against WRN or TRF1, respectively. Lane 1, input (4%); lane 2, precipitates with rabbit antiGFP (80%); lane 3, precipitates with rabbit IgG control (80%). (D) ChIP assay telomeric DNA recovery. Purified DNA from precipitated protein-DNA complexes were analyzed with a probe against telomeric DNA (upper), or Alu DNA (lower) by slot blot analysis. Lane 1, precipitates with rabbit anti-GFP (83%); lane 2, precipitates with rabbit IgG control (83%); lane 3, input (2%).

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Figure 4. WRN Helicase and Exonuclease Cooperate to Dissociate the Telomeric D Loop (A) Telomeric D loop schematic. A bubble was formed by annealing the BT and BB strands. The INV strand with a 5⬘ duplex end (hairpin) was hybridized in the melted region. Dotted lines indicate continuity in the DNA strand. RE sites (boxed) and cleavage sites (arrows) are shown. The four telomeric repeats are underlined and numbered. Nucleotide positions in INV starting from the 5⬘ end are indicated. (B) Annealing reactions contained either one strand, INV (lane 1) and BB (lane 4); two strands, INV/BB (lane 2) and BT/BB (lane 5); or three strands INV/BB/BT (lanes 3 and 6). Asterisk indicates the 5⬘ end-labeled strand. Substrates were run on an 8% native gel. (C) Digestion reactions contained a D loop with a labeled INV strand and either HpaI (lane 3) or FokI (lane 4). Products were run on a 14% denaturing gel. Lane 1, marker; lane 2, uncut D loop. Oligonucleotide sizes are indicated. (D and E) WRN protein was incubated with the D loop substrate containing a labeled INV strand (0.5 nM) for 15 min at 37⬚C. Reaction aliquots were run on an 8% native gel (D) and on a 14% denaturing gel (E). The reactions contained 0.75, 1.5, 3, and 6 nM WRN with either 2 mM ATP (lanes 1–5), 2 mM ATP␥S (S, lanes 6–9), or no ATP (lanes 11–14, [D]; lanes 10–13, [E]). ⌬, heat-denatured substrate. Numbers in (E) indicate product length. (F) Reactions contained the D loop (0.5 nM) and 0, 0.75, 1.5, 3, or 6 nM X-WRN (lanes 1–5, left) or the 22 bp forked duplex (0.5 nM) and 0 or 6 nM X-WRN (lanes 1 and 2, right). Substrates were run on an 8% (left) or 12% (right) native gel. ⌬S, heat-denatured substrate.

corresponded to digestion termination at the G-rich sites of the telomeric repeats (Figures 4A and 4E). WRN did not digest the blocked ends of the bubble or dissoci-

ate the duplex bubble arms, as tested using a D loop with a labeled BB strand (data not shown). Both WRN activities were required for release of the

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longer INV products. When the WRN helicase activity was eliminated by substitution with ATP␥S (Figure 4D, lanes 6–9) or ATP omission (Figure 4D, lanes 11–14), the released 58- and 52-mer products were absent. Instead, shorter fragments (ⱕ46 nt) were observed (Figure 4E), which likely resulted from substrate digestion to unstable duplex lengths (ⱕ12 bp). Digestion of the 67-mer INV strand to 58- and 52-mer products decreases the duplex length to 24 and 18 bp, respectively, which are thermally stable under the reaction conditions (data not shown). Thus, the appearance of dissociated 58- and 52-mer products was dependent on the WRN helicase activity. When the WRN exonuclease was inactivated by a point mutation (E84A, X-WRN variant), no disruption of the D loop was observed (Figure 4F), although a short forked duplex was unwound as observed previously (Opresko et al., 2001). Therefore, the WRN helicase and exonuclease cooperate to resolve the telomeric D loop in a process that requires shortening of the 3⬘ telomeric tail.

Telomere Binding Proteins Limit WRN Digestion of the Telomeric D Loop Unregulated WRN exonuclease activity at the 3⬘ end of the invading strand of a telomeric D loop would result in truncation or loss of the 3⬘ telomeric tail, thereby compromising telomere function. TRF2 protects the 3⬘ telomeric tail since expression of a TRF2 DN mutant results in loss of this tail (van Steensel et al., 1998). Therefore, we asked whether the telomere binding proteins could regulate WRN activity on the telomeric D loop. We first tested for TRF1 and TRF2 binding under the WRN reaction conditions. Incubation of the D loop (lane 1) with TRF1 (lanes 2–5) or TRF2 (lanes 6–9) shifted the substrate mobility in a native gel (Figure 5A). Nearly all the substrate was shifted at the higher amounts of protein, and at 45 nM TRF2 most of the complex was retained in the well. We previously observed that retention of TRF2-DNA complexes in the well was dependent on telomeric sequence (Opresko et al., 2002). In contrast, the telomeric D loop was not shifted upon incubation with the TRF2 DN mutant (45 nM), which lacks the DNA binding domain (Figure 5A, lane 12). Prebound TRF2 altered the digestion profile of the WRN 3⬘ to 5⬘ exonuclease on the D loop. TRF2 may recruit WRN to the D loop, since the percent of undigested substrate decreased from 24% ⫾ 6% to 6.1% ⫾ 0.7% in the presence of 45 nM TRF2 (Figure 5B). However, TRF2 simultaneously limited the extent of digestion by WRN. There was a 4- to 5-fold increase in products shortened a few nucleotides within the unique sequence (66–59 nt), in the presence of the equal molar (5.5 nM) and excess (45 nM) TRF2, respectively (Figure 5B). There was a concomitant 4- to 8-fold decrease in digestion that progressed through the 1st and 2nd telomeric repeats and terminated at the 3rd repeat, with equal molar and excess TRF2 (Figure 5B). TRF2 regulation of WRN activity was dependent on substrate interaction. Heat-inactivated TRF2 (Figure 5B, lane 8) and a GST-TRF2 DN mutant (lane 10) did not affect the WRN exonuclease. TRF1 also limited the extent of WRN digestion at the

3⬘ end of the INV strand, although to a lesser extent. Increasing TRF1 protein caused an increase (6.7- to 8.8fold) in substrate shortened a few nucleotides within the unique sequence (66–59 nt), with equal molar (5.5 nM) or excess (45 nM) TRF1 (Figure 5C). There was a concomitant 2- to 4.4-fold decrease in digestion that progressed through the 1st and 2nd telomeric repeats and terminated at the 3rd repeat, with equal molar and excess TRF1, respectively (Figure 5C). In contrast, TRF1 (45 nM) preincubation with a D loop containing scrambled telomeric sequence did not alter the WRN exonuclease activity (Supplemental Figure S3 on Molecular Cell’s website). Prebound TRF1 (50 nM) on a 34 bp telomeric fork duplex also did not effect the WRN exonuclease (data not shown), as shown previously for TRF2 (Opresko et al., 2002). Therefore, TRF1 and TRF2 limit the extent of WRN exonuclease progression specifically on a telomeric D loop. Limitation of WRN digestion by TRF2 and TRF1 decreased the percent of D loops with a duplex region short enough to be completely unwound by the WRN helicase. Native gel analysis showed a 3-fold decrease in the percent of dissociated D loops from 55% ⫾ 8% to 17% ⫾ 3% in the presence of 45 nM TRF2 (Figure 5D). WRN-mediated release of the longer 58-mer (24 bp duplex) and 52-mer (18 bp duplex) products was more evident in the presence of TRF2, while the 46-mer product was primarily absent, compared to WRN alone (Figure 5D). A percentage of the telomeric D loop incubated with TRF2 and WRN was retained in the well even after proteinase K treatment for unknown reasons. TRF1 also decreased the percent of WRN-dissociated D loops 3-fold from 69% ⫾ 16% to 23% ⫾ 8% (Figure 5E). Release of the 52-mer (18 bp duplex) was observed in the presence of TRF1. In summary, TRF2 and TRF1 limit the extent of WRN exonuclease activity on a telomeric D loop, so that release of longer INV strands is favored. WRN/RPA Helicase Complex Resolves TRF1 and TRF2 Prebound Telomeric D Loops RPA stimulates WRN unwinding of long duplexes partly by coating the partially unwound single strands (Opresko et al., 2001). RPA stimulated WRN to release the full-length INV strand, while the percent of shortened products (ⱕ58-mer) decreased as a function of RPA (4.2–32 nM) (Figure 6A) as confirmed by denaturing gel analysis (data not shown). Up to 65% ⫾ 1% of the labeled INV strand was released as the full-length 67-mer in the presence of WRN and 32 nM RPA (Figure 6A). RPA also promoted WRN unwinding of the duplex arms of the bubble, as indicated using a D loop with a labeled BB strand (Figure 6A). Thus, RPA stimulates WRN dissociation of the telomeric D loop, in a manner that limits digestion of the 3⬘ telomeric tail and promotes unwinding of the bubble. We next examined unwinding in the presence of TRF1 and TRF2. In contrast to RPA, TRF1 and TRF2 do not coat ssDNA (Broccoli et al., 1997) and are unable to promote WRN unwinding of long duplexes (34 bp) (Opresko et al., 2002). When sufficient concentrations of RPA (32 nM) were used for optimal unwinding by WRN, dissociation of the INV strand was not affected by prebound excess TRF2 or TRF1 (45 nM) (Figure 6B).

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Figure 5. TRF2 and TRF1 Limit the Extent of WRN Exonuclease Digestion on a Telomeric D Loop (A) Binding reactions contained 0.5 nM substrate (lane 1 and 10) and either TRF1 (5.5, 11, 22.5, and 45 nM, lanes 2–5), TRF2 (5.5, 11, 22.5, and 45 nM, lanes 6–9), GST protein (45 nM, lane 11), or the GST-TRF2 DN mutant (45 nM, lane 12). Products were run on a 5% native gel. The arrow indicates the well position, and the percent bound (%B) is shown at the bottom of each lane. (B and C) The D loop (0.5 nM) was preincubated with different concentrations of TRF2 (B) or TRF1 (C) (0–45 nM; monomer) for 5 min on ice prior to adding WRN. Products were run on a 14% denaturing gel. ⌬E, heat-inactivated protein (45 nM); GST, glutathione protein (45 nM); DN, GST-TRF2 DN mutant (45 nM). Numbers ⫽ oligonucleotide length. The percent of undigested substrate and products shortened to each length range (as defined by the telomeric repeats) were calculated (see Experimental Procedures) and plotted as shown: 0 nM, black bars; 5.5 nM, gray bars; 45 nM, hatched bars. (D) Products in (B) and (C) were run on an 8% native gel. ⌬S, heat-denatured substrate. %D represents the mean and standard deviation for the percent of total displacement (see Experimental Procedures).

The denaturing gel reveals limited digestion of the INV strand (Figure 6C). At suboptimal concentrations of RPA, so that WRN digestion products were apparent, we found that TRF2 still limited the extent of WRN digestion

(data not shown). Thus, RPA, TRF1, and TRF2 cooperate to promote WRN-mediated release of an intact 3⬘ telomeric tail from a D loop and limit the extent of trimming by the WRN exonuclease.

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Discussion We observed that WRN was in the majority of TRF1 foci that colocalized with PCNA replication foci in S phase ALT cells. In contrast, significant colocalization of WRN with PCNA or with TRF1 was not seen in S phase telomerase-positive HeLa cells. WRN associated directly with telomeric DNA in ALT cells. Thus, WRN appears to localize to telomeric ends when resolution of telomeric D loops is likely to be required for telomeric replication and/or recombination in telomerase-deficient cells. Consistent with this, the WRN helicase and exonuclease cooperated to dissociate a model telomeric D loop structure. TRF1 and TRF2 limited the extent of WRN exonuclease digestion of the D loop but did not block unwinding by a WRN/RPA complex. These results implicate WRN in processing of telomeric ends in telomerasedeficient cells. We demonstrated that WRN has the ability to resolve a telomeric D loop, which is not only a likely component of the telomere cap structure (Griffith et al., 1999) but is also an important recombination intermediate. Roles for RecQ helicases have been proposed for dissociating possible telomeric structure, such as D loops and/or G quadruplexes (Hickson, 2003; Opresko et al., 2003). Our studies do not exclude other potential substrates for WRN at the telomeres. However, we believe our observation that WRN associates with telomeric DNA in untreated ALT cells but not telomerase-positive cells strongly implicates telomere recombination intermediates as a likely substrate for WRN. The WRN helicase and exonuclease were shown to act independently on D loops and Holliday junctions (Orren et al., 2002; Constantinou et al., 2000; Mohaghegh et al., 2001). Our study constitutes a demonstration of the concerted and simultaneous action of the WRN helicase and 3⬘ to 5⬘ exonuclease in resolving a recombination intermediate. The exonuclease was required to shorten the duplex length and direct release of the invading strand by the helicase. Whether the WRN exonuclease and helicase coordinate in vivo is not known. However, the evolutionary conservation of these distinct activities on a common polypeptide argues for a cooperative function. Consistent with this, Chen et al. (2003) recently found that a balance between the WRN helicase and exonuclease activities was important for optimal repair by HR. Our findings may also extend to roles for the WRN helicase and exonuclease in resolving D loop intermediates in pathways outside the telomeres. The telomere binding proteins appear to regulate pro-

Figure 6. The WRN/RPA Helicase Complex Resolves Telomeric D Loops Prebound with TRF2 and TRF1 (A) WRN protein (6 nM) was incubated with a telomeric D loop containing a labeled INV strand (0.5 nM) and RPA (0, 4, 8, 16, and 32 nM) (lanes 2–6). RPA alone (32 nM), lane 8; ⌬S heat-denatured substrate, lane 7. WRN (6 nM) was also incubated with a D loop containing a labeled BB strand and either 0 nM (lane 11) or 32 nM (lane 12) RPA. BT/BB bubble substrate marker, lane 9; D loop marker, lane 10; ⌬S heat-denatured substrate, lane 13. Reactions

were incubated for 15 min at 37⬚C, and the products were run on an 8% native gel. The percent displacement of the full-length INV strand was quantitated (see Experimental Procedures) and plotted against RPA concentration. (B and C) The telomeric D loop (0.5 nM) with a labeled INV strand was preincubated with TRF2 or TRF1 (45 nM), as indicated, for 5 min on ice. Reactions were started by adding WRN (6 nM) with either 0 or 32 nM RPA, as indicated, and incubated for 15 min at 37⬚C. ⌬S heat-denatured substrate, lane 5. The reactions were run on an 8% native gel (B) and a 14% denaturing gel (C). The numbers indicate oligonucleotide size.

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cessing of telomeric DNA. A recent report found TRF2 recruited WRN to substrates with telomeric repeats and facilitated WRN exonuclease activity (Machwe et al., 2004). We observed a similar WRN recruitment to the telomeric D loop by TRF2 but not by TRF1 (Figures 5B and 5C). However, in our experiments TRF2 and TRF1 binding to the telomeric D loop limited the extent of WRN exonuclease digestion into the telomeric repeats. Discrepancies between these studies may be ascribed to differences in reaction conditions. Machwe et al. (2004) examined the exonuclease in the absence of helicase activity, whereas we examined the simultaneous contribution of both. Our findings are consistent with reports that TRF2 protects the telomeric tail in vivo and that the telomeric tail is lost upon expression of a TRF2 DN mutant (van Steensel et al., 1998). Furthermore, we observed that TRF2 and TRF1 did not block WRN-mediated dissociation of the D loop, as long as sufficient RPA was present to stimulate release of the full-length strand (Figure 6B). These results indicate that RPA, TRF1, and TRF2 cooperate to promote WRN-mediated release of an intact 3⬘ telomeric tail and limit processing of the 3⬘ end by the WRN exonuclease. Together, the biochemical and cellular data indicate that WRN likely plays a role at telomeres during S phase in untreated telomerase-deficient cells. The majority of TRF1 that colocalized with PCNA foci in S phase ALT cells also contained WRN (Table 1). This indicates that WRN may play a role in releasing the t loop by resolving the intratelomeric D loop to allow for DNA replication fork progression to the chromosome end. Interference with the replication fork may lead to DNA breaks at the telomeres, and telomerase could extend the broken telomeres to functional lengths. Alternatively, WRN may be important for dissociating the telomeric tail from aberrant and potentially toxic recombination intermediates. Once the t loop is dissociated during DNA replication, the 3⬘ single strand tail is released and can be engaged and protected by telomerase (Blackburn, 2000). In the absence of telomerase, the 3⬘ ssDNA tail is recombinogenic and could inappropriately invade homologous telomeric DNA or homeologous genomic DNA. This may occur more frequently in telomerasedeficient cells, including perhaps normal somatic cells, compared to telomerase-positive cells. Thus, WRN could prevent aberrant recombination by dissociating the intermediate. The ability of WRN to dissociate the telomeric tail from D loops may also be important for recombination repair of telomeric ends. AA-PML bodies most likely represent active sites of ALT rather than storage depots. These bodies not only contain proteins required for recombination (Yeager et al., 1999) but are enriched when HR is most active. AA-PML bodies also colocalize with BrdU incorporation (Wu et al., 2000, 2003), consistent with our results showing WRN colocalization with TRF1/PCNA foci in S phase ALT cells (Figure 2). WRN colocalizes with foci of average and high telomeric DNA content. The latter may represent telomeric clusters involved in recombination or extrachromosomal telomeric DNA, which are proposed to be used in ALT pathways (Henson et al., 2002). We also observed some WRN/TRF1 colocalization in the absence of PCNA (Table 1) and in non-S phase cells (Figure 2D). Thus, WRN may localize to telo-

meres prior to the replication fork and/or may function in telomere recombination repair outside of S phase. The ALT pathway may represent an unregulated variant form of a normal recombination repair process that restores broken or damaged telomeres and, thus, could prevent aberrant and untimely telomere uncapping. This may be particularly important in telomerase-deficient somatic cells, since telomerase can recap and extend broken telomeres (Blackburn, 2000). In the scenarios presented above, the WRN helicase acts to resolve recombination intermediates at telomeric ends. Toxic recombination intermediates may contribute to defects in WS cells (Prince et al., 2001). Expression of a bacterial resolvase RusA or suppression of recombination with a Rad51 dominant-negative mutant in WS cells significantly improved cell survival after damage (Saintigny et al., 2002). Unresolved recombination intermediates at the telomeres may lead to chromosomal breakage and/or end fusions. Consistent with this, WS fibroblasts show DNA rearrangements, translocations, and an increased frequency of dicentric chromosomes (Salk et al., 1985; Stefanini et al., 1989; Grigorova et al., 2000). Furthermore, expression of a WRN helicase-dead mutant in a telomerase-positive tumor cell line increased chromosome fusions and stochastic telomere loss (Bai and Murnane, 2003). Thus, even in telomerase-positive cells, WRN defects may lead to telomere loss and increased genomic instability. In summary, we propose that WRN may be involved both in recombination repair at telomeres and in the dissociation of a 3⬘ telomeric tail from inappropriate recombination intermediates by resolving telomeric D loops. Defects in these processes may contribute to telomere dysfunction, which could trigger premature exit from the cell cycle. The presence of active telomerase may compensate for repair processes that restore broken or damaged telomeres, since telomerase lengthens telomeres. Telomerase also engages and protects the 3⬘ telomeric tail and may prevent inappropriate strand invasion (Blackburn, 2000). These results may partially explain why the forced expression of exogenous telomerase in WS fibroblasts rescues the premature senescent phenotype (Wyllie et al., 2000). Experimental Procedures Proteins Restriction enzymes (RE) and T4 polynucleotide kinase (PNK) were purchased from New England BioLabs. Human RPA was generously provided by Dr. Mark Kenny (Albert Einstein Cancer Center, NY). For details in protein purification see Supplemental Data on Molecular Cell’s website. Recombinant histidine-tagged wild-type and mutant WRN proteins were purified using a baculovirus/insect cell expression system. Recombinant histidine-tagged human TRF2 and TRF1 protein was purified using a baculovirus/insect cell expression system as previously described for TRF2 (Opresko et al., 2002); the baculovirus constructs were generously provided by Dr. Titia de Lange (Rockefeller University, NY). The recombinant GST-TRF2 DN mutant was generated by PCR cloning. Procedures for the expression and purification in E. coli were as previously described (von Kobbe et al., 2003). Cloning of Fluorescent-Tagged Fusion Proteins EYFP-WRN and ECFP-PCNA were made as previously described (Baynton et al., 2003). HcRed-PCNA was made by switching the AgeI/MluI fragment (ECFP) from ECFP-PCNA with the correspond-

WRN Resolution of Telomeric D Loops 773

ing HcRed-C1 fragment (Clonetech). TRF1 was PCR amplified from the TRF1 cDNA and ligated into the XmaI/PstI restriction site of pECFP-C1 vector (Clonetech). All constructs were verified by DNA sequencing. EYFP-TRF1 was made by switching ECFP with EYFP from the EYFP-C1 vector (Clonetech) after AgeI/XhoI digestion. Cell Culture Cells were transfected with Fugene (Roche) (U-2 OS) or CaPO4⫺ method (Profection Promega) (HeLa) according to the manufacturer’s recommendations. YFP-TRF1/YFP-WRN expressing U-2 OS ALT cells were cultured in DMEM media containing fetal calf serum, penicillin, streptomycin, glutamine, and geneticin G418 (400 ␮g/ml, Invitrogen). Untransfected HeLa and U-2 OS cells were cultured in the same media without geneticin. Confocal Microscopy A Zeiss LSM 510 laser scanning microscope equipped with a PlanApochromate 63⫻/1.4 oil immersion objective was used to examine images of 1 ␮m thick slices of cycling living cells. Intensities were measured as follows. ECFP: excitation (ex) at ␭ ⫽ 458 nm, detection (det) at 475 nM ⬍ ␭ ⬎ 525 nM; EYFP: ex at ␭ ⫽ 488 nm, det at ␭ ⬎ 505–550 nm when together with HcRed/Cy3, otherwise ex at ␭ ⫽ 514 nm, det at ␭ ⬎ 560 nm; HcRed/Cy3: ex at ␭ ⫽ 543 nm, det at ␭ ⬎ 585 nm. Immunofluorescence and Q-FISH EYFP-TRF1 and EYFP-WRN expressing U-2 OS cells were cultured in slides. Immunofluorescent staining with a monoclonal mouse PML-antibody (#MS-1796, NeoMarkers) was performed using standard procedures (see Supplemental Data on Molecular Cell’s website). The slides were then stained using a Q-FISH kit with a telomerespecific CY3-PNA-probe (DAKO, Denmark), and the signals were quantitated according to the manufacturer (see Suplemental Data on Molecular Cell’s website for details). In the cell population displaying some WRN and telomere colocalization, the signal intensity was measured for each telomere (n ⫽ 1991), including those that colocalized with WRN (n ⫽ 222) and those that did not (n ⫽ 1769). The percent of EYFP-WRN colocalizing and noncolocalizing telomere foci at each signal intensity was calculated as a function of total telomere foci and plotted against the standardized signal intensity. Chromatin Immunoprecipitation ChIP assays were performed with U-2 OS cells that stably express EYFP-TRF1 or EYFP-WRN using a ChIP assay kit (Upstate) according to the manufacturer with some modification. Immunoprecipitation of EYFP-WRN and EYFP-TRF1 was achieved with rabbit polyclonal anti-GFP (5–7.5 ␮g) antibody against the EYFP variant (NB 600-303, Novus). For details see Supplemental Data on Molecular Cell’s website. Construction of the D Loop Oligonucleotides shown in Figure 4 were from Sigma-Genosys or from Lofstrand Laboratories and were gel purified by the manufacturer. Oligonucleotides BT and BB contain three phosphorothioated nucleotides at the 3⬘ end to block digestion by the WRN exonuclease. Oligonucleotides (INV or BT) were 5⬘ end labeled with [␥-32P]ATP (3000 Ci/mmol) with T4 PNK according to the manufacturer. Annealing reactions (20 ␮l) were conducted in 50 mM LiCl using a PC-100 Peltier thermal cycler (MJ Research). Labeled oligonucleotides (INV or BB) (4.5 fmol) were incubated with the complementary strand (INV or BB) (8 fmol) at 95⬚C for 5 min and cooled stepwise (1.2⬚C/min) to 60⬚C. Then oligonucleotide BT (10 fmol) was added and incubated at 60⬚C for 1 hr, followed by cooling stepwise (1.2⬚C/min) to 25⬚C. Substrates were analyzed on an 8% polyacrylamide native gel and visualized using a Phosphorimager (Molecular Dynamics). Substrates (100 fmol) were incubated with HpaI (2.5 U) or FokI (2 U) in 10 ␮l reactions for 4 hr at 37⬚C, according to the manufacturer. The reactions were analyzed on a 14% denaturing polyacrylamide gel and visualized by Phoshorimager. WRN Helicase and Exonuclease Reactions Reactions (30 ␮l) were performed in standard reaction buffer (Opresko et al., 2001), unless otherwise indicated. DNA substrate

and protein concentrations were as indicated in the figure legends. The reactions were initiated by WRN addition and incubated at 37⬚C for 15 min. A 10 ␮l aliquot was mixed with 10 ␮l formamide stop dye and analyzed on a 14% denaturing polyacrylamide gel. The remainder of the reaction (20 ␮l) was added to 10 ␮l of 3⫻ native stop dye supplemented with 75 ␮g/ml proteinase K and a 10⫻ molar excess of unlabeled competitor oligonucleotide (Opresko et al., 2002). Products were deproteinized for 30 min at 37⬚C, followed by analysis on an 8% native polyacrylamide gel. Products were visualized by Phosphorimager and were quantitated with ImageQuant software (Molecular Dynamics). For the exonuclease products, the percent of unused substrate and shortened products in specific size ranges (see Figure 5 legend) was calculated as a function of the total radioactivity in the reaction lane. For the helicase products, the percent of displaced products was quantitated as previously described (Opresko et al., 2001). All values were corrected for background in the no enzyme control lane. Reported values represent the mean and standard deviation from at least three independent experiments. Electrophoretic Mobility Shift Assay Binding reactions (20 ␮l) were performed in the identical buffer as for the WRN activity reactions. The D loop substrate (0.5 nM) was incubated with increasing protein as indicated in Figure 5A’s legend for 20 min at 4⬚C, followed by addition of 10 ␮l 3⫻ dye (0.125% bromophenol blue and 40% glycerol) on ice. Reactions were loaded on a 5% native polyacrylamide (37.5:1) gel and electrophoresed at 200 V for 2.5 hr in 1xTAE. Acknowledgments We thank Drs. Jeanine Harrigan, Rika Kusumoto, and Robert Brosh for critical reading of the manuscript. We also thank Drs. Cayetanno von Kobbe, Wen-Hsing Cheng, and Byung Chan Ahn for helpful discussions. Received: December 2, 2003 Revised: April 27, 2004 Accepted: April 27, 2004 Published: June 17, 2004 References Bai, Y., and Murnane, J.P. (2003). Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein. Hum. Genet. 113, 337–347. Baynton, K., Otterlei, M., Bjoras, M., von Kobbe, C., Bohr, V.A., and Seeberg, E. (2003). WRN interacts physically and functionally with the recombination mediator protein RAD52. J. Biol. Chem. 278, 36476–36486. Blackburn, E.H. (2000). Telomere states and cell fates. Nature 408, 53–56. Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., and Wright, W.E. (1998). Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352. Broccoli, D., Smogorzewska, A., Chong, L., and De Lange, T. (1997). Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat. Genet. 17, 231–235. Chen, L., Huang, S., Lee, L., Davalos, A., Schiestl, R.H., Campisi, J., and Oshima, J. (2003). WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair. Aging Cell 2, 191–199. Cohen, H., and Sinclair, D.A. (2001). Recombination-mediated lengthening of terminal telomeric repeats requires the Sgs1 DNA helicase. Proc. Natl. Acad. Sci. USA 98, 3174–3179. Constantinou, A., Tarsounas, M., Karow, J.K., Brosh, R.M., Bohr, V.A., Hickson, I.D., and West, S.C. (2000). Werner’s syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. EMBO Rep. 1, 80–84. Davis, T., Singhrao, S.K., Wyllie, F.S., Haughton, M.F., Smith, P.J.,

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