Report

Defective Telomere Lagging Strand Synthesis in Cells Lacking WRN Helicase Activity

See allHide authors and affiliations

Science  10 Dec 2004:
Vol. 306, Issue 5703, pp. 1951-1953
DOI: 10.1126/science.1103619

Abstract

Cells from Werner syndrome patients are characterized by slow growth rates, premature senescence, accelerated telomere shortening rates, and genome instability. The syndrome is caused by the loss of the RecQ helicase WRN, but the underlying molecular mechanism is unclear. Here we report that cells lacking WRN exhibit deletion of telomeres from single sister chromatids. Only telomeres replicated by lagging strand synthesis were affected, and prevention of loss of individual telomeres was dependent on the helicase activity of WRN. Telomere loss could be counteracted by telomerase activity. We propose that WRN is necessary for efficient replication of G-rich telomeric DNA, preventing telomere dysfunction and consequent genomic instability.

Werner syndrome (WS) patients suffer from multiple signs of premature aging (1). A wide variety of cells are affected, which suggests a housekeeping dysfunction. Cells from WS patients grow poorly in culture and enter senescence prematurely, phenotypes that can be reversed by expression of the catalytic telomerase subunit human telomerase reverse transcriptase (hTERT) (2), therefore implicating a critical role for telomere integrity in WS pathogenesis.

Because of their G-rich nature, telomeres have been suggested to contain energetically stable non-B form DNA such as G-quadruplexes. WRN, previously found to associate with ALT-associated PML bodies (3), has been suggested as capable of unwinding such structures as well as resolving telomeric D-loops in vitro (1, 3, 4). This suggests that the role of WRN may be related to its ability to resolve G-rich structures during DNA replication. To investigate the potential involvement of WRN in telomere replication, we expressed wild-type WRN and a putative dominant negative WRN allele with an inhibitory mutation in the helicase domain (K577M) (5, 6) in primary IMR90 fibroblasts and in HeLa cells. Fluorescent in situ hybridization (FISH) of metaphase telomeres (7) prepared from IMR90 fibroblasts and from HeLa cells expressing the helicase-deficient WRN allele revealed a number of chromosomes in which telomeric signals were only detected at a single chromatid, a phenotype we termed sister telomere loss (STL) (Fig. 1A). HeLa cells infected with a control virus or a virus expressing wild-type WRN displayed a low frequency of STL (0.8 events per cell) (8). STL increased significantly to 2.2 events per cell when the dominant negative WRN was expressed (Table 1). Consistent with telomerase alleviating the growth phenotypes in WS cells (2) and with the proposed preference of telomerase for critically short telomeres (9), inhibition of the catalytic subunit of telomerase (hTERT) (10, 11) with a specific drug (BIBR1532) (12, 13) led to a further increase of STL to 4.3 events per HeLa cell (Table 1). Expression of the helicase-deficient WRN allele in telomerase-deficient primary IMR90 fibroblasts also increased STL significantly to 4.1 events per cell (Table 1). Taken together, these data suggest that the helicase activity of WRN is required for proper telomere maintenance.

Fig. 1.

Dysfunctional telomeres in WS cells. (A) FISH analysis of metaphase chromosomes (8) of IMR90 WRN, IMR90 WRN-K577M, HeLa WRN-K577M, and AG05229C primary WS fibroblasts. Telomeres were hybridized with a TRITC-[TTAGGG]4 probe and are shown in the red channel; DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) and is shown in the blue channel. Arrows indicate missing telomeric staining from single sister chromatids. (B) An individual chromosome from an AG05229C WS fibroblast. (C) Western blot of WRN, WRN-E84A, and WRN-K577M in AG05229C WS fibroblasts. Antibodies to WRN and to γ-tubulin were used to detect the indicated proteins. (D) Quantification of cells showing staining with an antibody directed against ATM phosphorylated at serine 1981 (8). IMR90 fibroblasts infected with a control virus and primary WS cells were analyzed. Werner cells were infected with a control retrovirus or with viruses expressing WRN, nuclease-deficient WRN (E84A), or helicase-deficient WRN (K577M). Only cells with two or more prominent ATM foci were considered. (E) Anaphases from IMR90 cells and AG03141D and AG05229C Werner fibroblasts expressing HPV16 E6 and E7 oncoproteins. DNA has been stained with DAPI and is shown in blue.

Table 1.

Effect of WRN on sister telomere loss (loss of the telomeric signal from one of two sister chromatids). At least 26 metaphases per cell line were analyzed. P values were determined by Wilcoxon two-sided ranks test analysis.

Cell line STL/cell Significance
HeLa control 0.8
HeLa WRN 0.9
HeLa WRN K577M 2.2 P < 0.0001
HeLa BIBR controlView inline 1.1
HeLa BIBR WRN 1.0
HeLa BIBR WRN K577M 4.3 P < 0.0001
IMR90 0.8
IMR90 WRN K577M 4.1 P < 0.0001
AG03141D control 2.8
AG05229C control 1.7
AG05229C WRN 1.2 P < 0.0001
AG05229C WRN E84A 1.1 P < 0.0001
AG05229C WRN K577M 2.0 P < 0.0001
AG05229C hTERT 0.4 P < 0.0001
  • View inline* Cells were treated with BIBR 1532 for 2 days before analysis.

  • To test genetically the requirement of WRN for telomere stability, we looked for STL in primary fibroblasts from WS patients. Chromosomes with missing telomeres on single chromatids were readily detected in WS fibroblasts from the AG03141D and AG05229C cell lines (Fig. 1A), at a frequency of 2.8 and 1.7 events per cell, respectively (Table 1). No preference for p- or q-arm STL was observed, because single signals on both arms could be observed (Fig. 1B). Reconstitution of the AG05229C cells with wild-type WRN, a nuclease-deficient WRN-E84A allele (14), or helicase-deficient WRN-K577M (Fig. 1C) further supported the requirement of WRN helicase activity for telomere maintenance. Expression of WRN and WRN-E84A reduced the frequency of STL to a background level of 1 event per cell, whereas expression of WRN-K577M failed to reduce STL frequencies (Table 1). Expression of the catalytic subunit of telomerase markedly reduced the occurrence of STL (Table 1).

    Excessive loss of telomeric DNA leads to induction of the DNA damage machinery (1517). Twenty-eight percent of primary WS fibroblasts stained positively with an antibody directed against ataxia telangiectasia mutated (ATM) phosphorylated at serine 1981 (phospho-ATM) (Fig. 1D) (18). In comparison, despite a higher S phase index, only 6% of IMR90 fibroblasts showed such staining. Expression of wild-type WRN and nuclease-deficient WRN reduced the percentage of phospho-ATM–positive cells to 17% and 17.5% (P < 0.0001), respectively. Helicase-deficient WRN did not alter the percentage of cells with phospho-ATM foci, suggesting that the helicase activity of the protein is required to suppress the initiation of DNA damage signals. Expression of hTERT in WS fibroblasts, however, almost completely repressed ATM foci formation (Fig. 1D and fig. S1), consistent with our finding that telomerase expression lowers the frequency of STL (Table 1). Collectively, our data suggest that lack of WRN helicase activity leads to DNA damage signaling, likely because of telomere loss on single sister chromatids.

    To establish whether STL leads to chromosome fusions, WS cells were analyzed for DNA bridges during anaphase (19). When the pRB and p53 pathways were suppressed by expression of the human papilloma virus serotype 16 (HPV16) E6 and E7 oncoproteins, the number of chromosome fusions, seen as anaphase bridges, was sharply elevated (Fig. 1E and table S1). Fusions were maintained in the checkpoint-suppressed cells, independently of the expression of WRN alleles. hTERT expression lowered the frequency of anaphase bridges by 75%, which stresses the importance of telomerase in alleviating defects in WS cells, as well as in telomerase-suppressed HeLa cells (table S1).

    STL points to a dysfunction in telomere replication during S phase. Telomeric chromatin immunoprecipitation (ChIP) assays (8, 20) with an antibody to WRN in primary IMR90 fibroblasts led to recovery of TTAGGG repeats in extracts from S phase cells (recovery of 5.5% of input telomeric DNA). No signal was detected in extracts from G2, M, and G1 cells. The S phase–specific association of WRN with telomeres was not due to cell cycle–regulated levels of WRN protein amounts (Fig. 2A). An alternative assessment of the association of WRN with telomeres, using immunostaining analysis (8) in IMR90 fibroblasts, uncovered similar results (fig. S2). In contrast, the duplex telomere-binding proteins TRF1 and TRF2 were associated with TTAGGG repeats throughout the cell cycle (Fig. 2, A and B). Thus, WRN has the ability to associate with telomeres and does so preferentially during the DNA synthesis phase of the cell cycle.

    Fig. 2.

    S phase–specific telomeric association of WRN in primary human IMR90 fibroblasts. (A) ChIPs on IMR90 cells over the cell cycle. Immunoprecipitations were performed with the indicated antibodies and dot blots hybridized with TTAGGG and ALU probes (8, 20). Fractions of cells in the according cell cycle phase are indicated in percentage. The same extracts were used for WRN and γ-tubulin Western blotting. Quantification of the Western blot is indicated in percentage of the S phase signal. IgG, immunoglobulin G. (B) Quantification of the data in (A), n = 6 independent ChIP experiments.

    On the basis of our findings that WRN localizes to telomeres in primary cells in S phase and that WS cells show telomerase-dependent catastrophic loss of telomeres from single sister chromatids, we considered the possibility that WRN played a role in telomere replication. Telomeres replicated by either leading or lagging strand synthesis can be distinguished by chromosome orientation FISH (CO-FISH) (21) (Fig. 3A). HeLa 1.2.11 cells, which carry telomeres with an average length of 20 kb (19), showed clear signals after double staining with a fluorescein isothiocyanate (FITC)–coupled peptide nucleic acid probe specific for the G strand and a tetramethyl rhodamine isothiocyanate (TRITC)–coupled probe specific for the C strand (Fig. 3B). Inhibition of WRN by the dominant negative helicase-deficient allele led to the loss of single sister telomeres (Table 1), and this loss almost exclusively affected lagging strand telomeres (Fig. 3B, loss of green signal). The frequency of lagging strand sister telomere loss increased from 0.5 to 3.6 events per cell, after expression of the helicase-deficient WRN allele (Fig. 3C, P < 0.0001), whereas the frequency of leading strand telomere loss was unaffected.

    Fig. 3.

    Loss of lagging strand telomeres in WS cells. (A) Schematic of CO-FISH. Leading and lagging strand synthesis incorporates bromodeoxyuridine and bromodeoxycytidine (BrdC) into freshly synthesized DNA strands. Bromo-nucleotide–substituted DNA is degraded, and telomeres are hybridized with FITC-[CCCATT]4 and TRITC-[TTAGGG]4 probes (8). Currently, the CO-FISH technique can be used successfully only on cells with long telomeres (>15 kb), which excludes WS cells from the analysis. (B) CO-FISH of control HeLa 1.2.11 cells and HeLa 1.2.11 cells expressing WRN-K577M. Leading strand telomeres are shown in red and lagging strand telomeres in green. Cells were treated with the telomerase inhibitor BIBR1532 (Boehringer) 2 days before analysis. The arrows indicate missing sister telomeres. (C) Quantification of (B).

    Collectively, these data argue that WRN is required for efficient and complete lagging strand replication of the G-rich telomeric strand. G-quadruplexes, likely to form in this region (22, 23), are potentially resolved by WRN, allowing the replication fork to progress through and replicate the telomeres completely. A possible interpretation is that lack of WRN helicase activity leads to stalling of telomere lagging strand synthesis, once the replication fork encounters structures it cannot bypass, explaining why Werner cells enter senescence with telomeres generally longer than control cells (24). As a result, the exposed single-stranded G-rich strand is degraded by an unspecified nuclease, leading to partial or complete loss of the few affected telomeres, a hypothesis that elucidates why analysis of individual telomeres using a G overhang–dependent technique failed to detect STL (25). Our model predicts that G-rich obstacles could occur not only at the base but also throughout the duplex TTAGGG region, leading to partial telomere loss. This is supported by the high frequency of unequal sister telomere staining intensity observed in aged WS cells (fig. S3). The existence of redundant activities that can resolve such structures is likely, and recently such a protein has been described in the mouse (26). By linking telomere loss with replication, the model presented here differs from previously suggested break repair- and recombination-dependent hypotheses for telomere loss in WRN-K577M–expressing cells (6).

    Our model also explains why expression of telomerase rescues growth deficiency and STL. Telomerase, preferentially acting on short telomeres (9, 27), could elongate affected chromosome ends, reducing the frequency of STL and reducing DNA damage signaling. This hypothesis is supported by the recent finding that a combined deletion of WRN and Terc in mice resembles WRN pathogenesis in humans (28). Our data provide a link between telomere dysfunction and the progeriatric disease WS and offer an explanation of why lack of WRN affects a wide number of cell types. It remains to be seen whether the genome instability observed in WS cells is due to telomere dysfunction.

    Supporting Online Material

    www.sciencemag.org/cgi/content/full/306/5703/1951/DC1

    Materials and Methods

    SOM Text

    Figs. S1 to S3

    Table S1

    References and Notes

    References and Notes

    View Abstract

    Navigate This Article