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RTEL1 Is a Replisome-Associated Helicase That Promotes Telomere and Genome-Wide Replication

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Science  11 Oct 2013:
Vol. 342, Issue 6155, pp. 239-242
DOI: 10.1126/science.1241779

RTEL1 in DNA Replication

Genome stability requires the coordinate action of a variety of DNA maintenance systems. The DNA helicase, RTEL1 (regulator of telomere length 1), disassembles recombination intermediates to avoid dangerous by-products. RTEL1 also limits excessive meiotic crossing over and disassembles telomere T loops. Vannier et al. (p. 239) now show that mammalian RTEL1 is part of the DNA replication machinery. RTEL1 binds to proliferating cell nuclear antigen (PCNA), an interaction that was important for normal DNA replication, replication fork stability, and telomere stability. The RTEL1-PCNA interaction was also critical for protecting cells against tumorigenesis but was not required for telomere T-loop disassembly.

Abstract

Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles telomere loops (T loops) and suppresses telomere fragility to maintain the integrity of chromosome ends. We established that RTEL1 also associates with the replisome through binding to proliferating cell nuclear antigen (PCNA). Mouse cells disrupted for the RTEL1-PCNA interaction (PIP mutant) exhibited accelerated senescence, replication fork instability, reduced replication fork extension rates, and increased origin usage. Although T-loop disassembly at telomeres was unaffected in the mutant cells, telomere replication was compromised, leading to fragile sites at telomeres. RTEL1-PIP mutant mice were viable, but loss of the RTEL1-PCNA interaction accelerated the onset of tumorigenesis in p53-deficient mice. We propose that RTEL1 plays a critical role in both telomere and genome-wide replication, which is crucial for genetic stability and tumor avoidance.

The stability of the genome is critically dependent on the coordinate action of DNA repair pathways during the cell cycle (1). The DNA helicase regulator of telomere length 1 (RTEL1) is an anti-recombinase that dismantles D-loop recombination intermediates to counter toxic DNA repair (2). RTEL1 also functions in meiosis to limit excessive crossing over (3) and disassembles T loops and suppresses telomere fragility to maintain integrity of the chromosome end (4). The mechanism(s) by which RTEL1 executes its function at sites of DNA repair and at telomeres remains unclear. Because RTEL1−/− mice are embryonic lethal (5) and die mid-gestation, it is also possible that RTEL1 possesses other essential functions that remain to be defined.

To further investigate RTEL1 function in cells, we performed mass spectrometry to identify interacting proteins from stable cells expressing a C-terminal green fluorescent protein (GFP)–tagged RTEL1. In addition to methyl methanesulfonate sensitive 19 (MMS19) that functions in Fe-S cluster assembly (6), we detected numerous components of the replisome, including replication factor C (RFC), DNA polymerases, minichromosome maintenance proteins (MCMs), and proliferating cell nuclear antigen (PCNA) (fig. S1A). We further investigated a putative RTEL1-PCNA interaction as bioinformatic analysis revealed the presence of a putative PCNA interaction motif (PIP box) located in the C terminus of the RTEL1 protein (fig. S1B). RTEL1-v5 and endogenous PCNA coimmunoprecipitated from cell extracts, and this interaction was resistant to benzonase treatment, excluding a nonspecific association via DNA bridging (Fig. 1A). By contrast, RTEL1-v5 proteins harboring five PIP box substitution mutations failed to immunoprecipitate PCNA (fig. S1C). The RTEL1-PCNA interaction is likely direct as in vitro pull-down experiments with a glutathione S-transferase (GST) fusion to a fragment of RTEL1 (amino acids 1125 to 1225) containing the wild-type PIP box efficiently bound to recombinant PCNA (rPCNA) as well as endogenous PCNA from human embryonic kidney–293 (HEK293) or HeLa cell extracts, whereas a GST-RTEL1 fusion harboring an I1169A PIP box mutant (referred to as IA) failed to interact (Fig. 1B). Arrayed biotinylated peptides comprising the wild-type RTEL1 PIP box sequence bound to rPCNA, whereas the RTEL1 PIP box mutant peptides did not (fig. S1D). These results demonstrate that the PIP box in RTEL1 confers a direct interaction with PCNA in vitro and in cells.

Fig. 1 RTEL1-PCNA interaction is dependent on RTEL1 PIP motif.

(A) Immunoprecipitation of RTEL1-v5 in ES cells stably overexpressing RTEL1-v5. IgG, immunoglobulin G; WB, Western blot. (B) GST pull-down experiments between wild-type (WT) RTEL1 or IA RTEL1 mutant and recombinant PCNA (rPCNA) extracts from HEK293 or from HeLa cells. (*) Truncated protein. (C) Detection of RTEL1-v5 (red) and PCNA (green) in ES cells overexpressing WT or IA RTEL1-v5 and PCNA-flag. DAPI, 4′,6-diamidino-2-phenylindole. (D) Immunoprecipitation (IP) of RTEL1-v5 and RTEL1-IA-v5 from ES cells derived from RTEL1+/+v5 and RTEL1IA/IAv5 mice.

PCNA is a processivity factor for DNA polymerase and an integral component of the replisome during S phase. PCNA is present in multiple discrete replication foci in S-phase cells (Fig. 1C) (7). Wild-type RTEL1-v5 also formed discrete foci, a subset of which colocalized with PCNA-Flag in unperturbed cells (Fig. 1C). The RTEL1 IA PIP box mutant is expressed in the nucleus, but failed to colocalize with PCNA (Fig. 1C). Thus, the presence of RTEL1 within replication foci is dependent on a PIP box–mediated interaction with PCNA. This result also suggests that any additional contacts between RTEL1 and other replisome components are likely to be weak or indirect, as they are not sufficient to maintain RTEL1 at replication foci when the RTEL1-PCNA interaction is abolished.

To investigate the functional importance of the RTEL1-PCNA interaction in vivo, RTEL1 IA PIP mutation marked with a v5 epitope was knocked into the RTEL1 locus in mice (fig. S2). The resultant mouse allele, termed RTEL1IA/IAv5, abolished RTEL1-PCNA binding as compared to the control mouse allele (RTEL1+/+v5), which retains an intact RTEL1 PIP motif (Fig. 1D). Loss of the RTEL1-PCNA interaction induced growth arrest and senescence in primary mouse embryonic fibroblasts (MEFs) derived from embryonic day 14.5 (E14.5) RTEL1IA/IAv5 embryos when compared to RTEL1+/+v5 control MEFs (Fig. 2A and fig. S3A), which we attribute to increased levels of spontaneous DNA damage in the mutant cells (fig. S5, E and F). Cell cycle analysis revealed a significant reduction in 5-bromo-2′-deoxyuridine (BrdU) incorporation and an accumulation of cells in late S/G2 in primary RTEL1IA/IAv5 MEFs when compared to control, suggestive of a defect in DNA replication (Fig. 2B and fig. S3B). Indeed, measurement of the progression of sister replication forks by molecular combing revealed that loss of the RTEL1-PCNA interaction caused forks to move considerably more slowly in a genome-wide manner compared to control cells (0.87 and 1.8 kb min−1, respectively; Fig. 2C). Of the replication forks, 52.9% were highly asymmetrical in RTEL1IA/IAv5 cells, compared to 7.8% asymmetric tracts in RTEL1+/+v5 control cells (Fig. 2, D and E), which suggests that loss of the RTEL1-PCNA interaction in cells results in increased replication fork stalling and/or collapse. Interorigin distances were also significantly shorter in the RTEL1IA/IAv5 cells when compared to control cells (87 ± 28 and 122 ± 28 kb, respectively; Fig. 2F), indicative of increased origin firing in mutant cells. Similar replication defects were also observed after conditional inactivation of RTEL1 following adenovirus-Cre treatment of RTEL1F/F MEFs (fig. S3, C to F). Blocking replication origin firing in the RTEL1IA/IAv5 cells with the CDC7 inhibitor PHA-767491 (fig. S4A) (8, 9) rescued both interorigin distance and replication fork extension rates to wild type but failed to rescue fork asymmetry (fig. S4, B to D). This suggests that increased origin usage and the reduction in fork speeds in RTEL1IA/IAv5 cells occur as a secondary consequence of a defect in preventing fork stalling and/or collapse. We propose that in the absence of the RTEL1-PCNA interaction, increased fork stalling and/or collapse or a failure to repair and/or restart a subset of replication forks triggers dormant origin firing, which in turn leads to a global reduction in replication fork extension rates.

Fig. 2 RTEL1-PCNA interaction promotes normal genome replication.

(A) Quantification of growth cell arrest after consecutive passages (P1 to P9) of each genotype. (B) BrdU incorporation of RTEL1IA/IAv5 and RTEL1+/+v5 primary MEFs. (C) Replication fork dynamics in RTEL1IA/IA and control MEFs pulse-labeled with iododeoxyuridine (IdU) or chlorodeoxyuridine (CldU) and subjected to DNA combing. One hundred fibers were measured per genotype, and replication fork speed was measured in kb min−1. (D) Representative images of sister replication forks in RTEL1+/+v5 and RTEL1IA/IAv5 primary MEFs. Arrows mark unstable and/or stalled forks. (E) Quantification of fork asymmetry in RTEL1IA/IAv5 and control primary MEFs. (F) Representative images and quantification of inter-origin distances (kb) in RTEL1IA/IAv5 and RTEL1+/+v5. Fifty fibers were measured per genotype. ****P < 0.0001 (two-tailed Mann-Whitney test). Error bars show the SD.

RTEL1 is proposed to maintain telomere integrity in part by catalyzing T-loop disassembly during S phase (4). Failure to dismantle T loops after inactivation of RTEL1 is associated with rapid telomere shortening and loss of the T loop as a circle (4). Phi29 polymerase–dependent telomere circles accumulate in cells after conditional inactivation of RTEL1 in RTEL1F/F MEFs (Fig. 3A) (4). By contrast, telomere circles (TCs) were undetectable in either RTEL1+/+v5- or RTEL1IA/IAv5-complemented embryonic stem (ES) cells (Fig. 3A). We also found no evidence of telomere loss in metaphase spreads of RTEL1+/+v5 or RTEL1IA/IAv5 cells (Fig. 3, B and C). RTEL1+/+v5 or RTEL1IA/IAv5 cells also exhibited a wild-type telomere length distribution (fig. S5A) and did not show any evidence of DNA damage at telomeres (fig. S5B), increased sister chromatid exchanges (SCEs), or increased telomere SCEs (fig. S5, C and D). Because RTEL1IA/IAv5 cells do not exhibit a significant induction of DNA damage at telomeres (fig. S5B), the elevated spontaneous levels of the histone γH2AX in these cells (fig. S5, E and F) are not caused by dysfunctional telomeres. These results establish that the RTEL1-PCNA interaction is dispensable for T-loop disassembly and preventing loss of the telomere as a circle.

Fig. 3 RTEL1-PCNA interaction is required to suppress telomere fragility but is dispensable to T-loop disassembly.

(A) Phi29-dependent TCs amplification and quantification in RTEL1+/+, RTEL1−/−, and ES cells complemented with RTEL1-v5 (+/+) and RTEL1-IA-v5 (IA/IA). (B) Quantification of telomere loss and fragile telomeres per metaphase in RTEL1+/+, RTEL1−/−, and ES cells complemented (Comp.) with RTEL1-v5 (Wt) and RTEL1-IA-v5 (IA/IA). (C) Representative images and quantification of fragile telomeres (arrow) and telomere loss (*) per metaphase in RTEL1+/+v5 and RTEL1IA/IAv5 primary MEFs subjected to the indicated treatments (–, no treatment; APD, aphidicolin; statistical analysis with two-tailed Mann-Whitney test). Error bars show the SD.

RTEL1 is also responsible for the suppression of telomere fragility (4, 10). Loss of RTEL1 in ES cells resulted in enhanced telomere fragility (24 ± 10 fragile telomeres per metaphase, compared to 8.8 ± 3.6 and 9.9 ± 4.2 in wild-type ES cells and RTEL1−/− ES cells complemented with RTEL1+v5; Fig. 3B and table S1). RTEL1−/− ES cells complemented with RTEL1IAv5 PIP box mutant and RTEL1IA/IAv5 MEFs exhibited high levels of telomere fragility (20 ± 7.9 and 10 ± 4.9, respectively; Fig. 3, B and C, and table S1), comparable to those detected in RTEL1-null ES cells or RTEL1−/− MEFs [24 ± 10 and (4), respectively]. After conditional inactivation of RTEL1 in RTEL1F/F MEFs, telomere fragility is exacerbated by treatment with aphidicolin or the G4-DNA stabilizer TMPyP4 (4). Similarly, treatment of RTEL1IA/IAv5 MEFs with aphidicolin or TMPyP4 resulted in a significant increase in telomere fragility, corresponding to 15 ± 5.5 and 14 ± 6.2 fragile telomeres per metaphase compared to 10 ± 4.9 fragile telomeres per metaphase without treatment (Fig. 3C and table S1). Collectively, these results establish that the RTEL1-PCNA interaction is essential for suppressing telomere fragility but is dispensable for T-loop disassembly (fig. S5G).

RTEL1-deficient cells are sensitive to G4-DNA stabilizer TMPyP4 (11) and telomere fragility is exacerbated by telomeric G4-DNA stabilization (4), suggesting a strong correlation between fragile telomeres and G4-DNA secondary structures. To address whether RTEL1 can disassemble telomeric G4-DNA, recombinant RTEL1 wild-type and adenosine triphosphatase (ATPase) dead (RTEL1 K48R) mutant proteins were purified and tested for activity toward telomeric G4-DNA structures. RTEL1 wild-type efficiently disassembled telomeric G4-DNA structures in an ATPase-dependent manner in vitro (fig. S6A). Addition of recombinant PCNA to the reaction neither stimulated nor inhibited the reaction. However, addition of TMPyP4 to the reaction inhibited the ability of RTEL1 wild type to disassemble the substrate (fig. S6B), suggesting that TMPyP4 exacerbates telomere fragility in vivo by blocking the ability of RTEL1 to dismantle telomeric G4-DNA structures.

RTEL1 was recently implicated in Hoyeraal-Hreidarsson syndrome, a severe form of the cancer predisposition and bone marrow failure syndrome dyskeratosis congenita (1214). A genome-wide association study also identified RTEL1 as a susceptibility locus for glioma (15, 16), raising the possibility that RTEL1 may function as a tumor suppressor. Despite the genome-wide replication problems in primary cells, RTEL1IA/IAv5 mutant mice are born at expected Mendelian ratios and exhibit normal body weight and morphology at adult stage. The viability of these mice is potentially explained by increased origin usage that likely compensates for the replication defects in cells. To examine the role of the RTEL1-PCNA interaction during tumor development, we established a cohort of compound Trp53−/−RTEL1+/IAv5 and Trp53−/−RTEL1IA/IAv5 mutant mice and monitored their survival. Homozygous Trp53−/−RTEL1IA/IAv5 mice exhibited substantially shorter life spans compared with control Trp53−/−RTEL1+/IAv5 mice (P < 0.0002; Fig. 4A). Tumor formation occurred in the Trp53−/−RTEL1IA/IAv5 mice within 115 ± 31 days compared with 147 ± 35 days for Trp53−/−RTEL1+/IAv5 mice. Histological analysis showed that both cohorts of Trp53−/−RTEL1+/IAv5 and Trp53−/−RTEL1IA/IAv5 mutant mice developed predominantly lymphomas (61.5 and 70.6%, respectively; Fig. 4B), although sarcomas and teratomas were also observed (15.4 versus 14.7% and 10.3 versus 14.7%, respectively; Fig. 4B), as described previously in Trp53−/− mice (17). Telomere analysis of the tumors from Trp53−/−RTEL1IA/IAv5 mice revealed extensive sister chromatid fusions, end-to-end fusions with telomeric repeats, and a high frequency of fragile telomeres when compared to nontumor cells from the same mice (Fig. 4, C and D, and fig. S7I). Furthermore, tumors from Trp53−/−RTEL1+/IAv5 mice or Trp53−/−RTEL1+/+ mice exhibited no evidence of telomere fragility or dysfunction (Fig. 4, C and D). Trp53−/−RTEL1IA/IAv5 mutant mice also developed medulloblastomas (12.8%; Fig. 4B and fig. S7, A to F), which were not observed in the Trp53−/−RTEL1+/IAv5 mice or in Trp53−/− mice described previously (17). Because RTEL1 is expressed in the cerebellar external granular layer (EGL) cells (fig. S7, G and H), which are the source of medulloblastomas (18), these data suggest that the RTEL1-PCNA interaction is important for protecting these EGL cells from transformation.

Fig. 4 RTEL1IA/IA mutation accelerates tumorigenesis in mice.

(A) Kaplan-Meier survival curves of Trp53−/−RTEL1IA/+v5 and Trp53−/−RTEL1IA/IAv5 mice with the number of mice indicated for each genotype. (B) Tumor spectrum in RTEL1IA/+v5 and RTEL1IA/IAv5 mice in Trp53−/− background. (C) Frequency of fragile telomeres and chromosome fusions in Trp53−/−RTEL1IA/IAv5 tumorigenic and nontumorigenic tissues. P value was calculated with two-tailed Mann-Whitney test. (D) Representative images of metaphase spreads derived from Trp53−/−RTEL1IA/+v5 and Trp53−/−RTEL1IA/IAv5 tumors subjected to telomere fluorescence in situ hybridization (red, telomeres; blue, DNA). White asterisk (*), sister chromatid fusions; yellow asterisk, end-to-end fusions; arrows, fragile telomeres.

Our data reveal a critical interaction between RTEL1 and PCNA that is essential for telomere and genome-wide replication and suppression of tumorigenesis in vivo. Suppression of telomere fragility, which we attribute to the telomeric G4-DNA unwinding activity of RTEL1, is absolutely dependent on the RTEL1-PCNA interaction. This suggests that replisome association is required for RTEL1 to counteract telomeric G4-DNA structures that arise at the replication fork. By contrast, the role for RTEL1 in T-loop disassembly and the suppression of telomere loss as a circle does not require the RTEL1-PCNA interaction. The RTEL1-PCNA interaction is also necessary to prevent replication fork stalling and/or collapse, which affects genome-wide replication. Finally, the accelerated rate of tumor formation and predisposition to medulloblastomas conferred by the RTEL1IA/IAv5 mutation in the Trp53−/− background implicates RTEL1 as a tumor-suppressor gene.

Supplementary Materials

www.sciencemag.org/content/342/6155/239/suppl/DC1

Materials and Methods

Figs. S1 to S7

Table S1

References (1926)

References and Notes

  1. Acknowledgments: We thank the Protein Analysis and Proteomics group at London Reasearch Institute for mass spectrometry and J. Mendez for MCM2 antibody. Research in the DNA damage response lab of S.J.B. is funded by Cancer Research UK and by a European Research Council (ERC) Advanced Investigator Grant (RecMitMei). S.J.B. is a recipient of a Royal Society Wolfson Research Merit Award. The laboratory of H.D. is supported by Canada Research Chair program, Canada Institute of Health Research, Manitoba Institute of Child Health, and Terry Fox Research Institute. J.-B.V. is funded by a long-term fellowship from ERC, and S.S. and Z.N. are supported by the fellowships from Natural Sciences and Engineering Research Council of Canada (to S.S.) and the Manitoba Health Research Council (to Z.N.).
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