RTEL1 contributes to DNA replication and repair and telomere maintenance

Abstract

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.

FIGURE 1:

Generation and characterization of epitope-tagged and fluorescently tagged mRtel1 knock-in ESCs. (A) Schematics of the mRtel1 locus, gene-targeting construct, and targeted locus. The top of the scheme depicts ∼36.6 kb of the mouse mRtel1 locus. Exons are indicated as black boxes. The second line represents the mRtel1-tag targeting construct. Using recombineering, we inserted a multipurpose tag directly 3′ of the gene in front of the termination codon in exon 34. The neomycin resistance marker surrounded by LoxP sites is located 3′ of the mRtel1 3′ UTR. The mutated genomic locus containing the mRtel1-tag fusion is shown at the bottom. The 3′ external probe and diagnostic EcoRV (EV) sites are indicated. Genotyping primers are indicated as arrows at the bottom. (B) Southern blot of EcoRV-digested genomic DNA of mRtel1^+/+ and mRtel1^+/tag+Neo knock-in ESCs. Wild-type and targeted mouse ESCs show the predicted restriction fragments using the probe indicated in A. (C) Immunoblots of mRtel1^+/+ and mRtel1^+/tag knock-in ESCs total protein extracts and (D) cytoplasmic and nuclear fractions. mRtel1-tag fusion protein was detected using an anti-FLAG antibody. (E) Fluorescence confocal and transmission images of C-terminally tagged mRtel1 in living mRtel1^tag/tag knock-in ESCs.

FIGURE 2:

mRtel1 is required during S phase. (A, B) Sensitivity of wild-type (C1) and two mRtel1-deficient (A4 and F2) ESC lines to the indicated doses of aphidicolin (A) and HU (B). Bars, mean percentage values of four experiments with SDs. (C) Analysis of the number of γ-H2AX foci per nucleus in wild-type (C1) and mRtel1-deficient (F2) ESCs. Foci in 1200 wild-type and 750 mRtel1-deficient nuclei in one focal plane were analyzed. (D) Images from a time-lapse movie of mRtel1-tag-DD expressed in mRtel1-deficient (F2) ESCs and cultured in the presence of aphidicolin (2 μM) and shield-1 (1 μM), both added at t = 0. Three image z-stacks (2.5-μm spacing) were acquired every hour. Projections of three z-stacks are depicted. Scale bar, 10 μm. (E) Colocalization of mRtel1-tag-DD and Cherry-m53BP1-M. Cells were cultured in the presence of aphidicolin (2 μM) and shield-1 (1 μM), and images depicted were taken at 17.5 h. Scale bar, 5 μm.

FIGURE 3:

mRtel1 promotes DNA repair. (A) Sensitivity of wild-type (C1) and two mRtel1-deficient (A4 and F2) ESC lines to the indicated doses of MMC. (B) mRtel1-tag-DD colocalizes with FancD2. Cells were cultured in the presence of shield-1 (1 μM) and 1 μg/ml MMC for 24 h and paraformaldehyde fixed. FandD2 was detected by immunofluorescence (ab2187; Abcam). Scale bar, 5 μm. (C, D) Sensitivity of wild-type (C1), two mRtel1-deficient (A4 and F2), and mRad54-deficient ESC lines to the indicated doses of γ-rays (C) and MMS (D). (E) Sensitivity of wild-type (C1), mRtel1-deficient (A4 and F2), and mRad17^{5′∆/5′∆} ESC lines to the indicated doses of UV. Bars, mean percentage values of four experiments with SDs.

FIGURE 4:

mRtel1 is important for HR. (A) Percentage of spontaneous (black bars) and MMC (0.2 μg/ml, 1 h) induced (white bars) SCEs per metaphase measured in wild-type (C1) and two mRtel1-deficient (A4 and F2) ESC lines. Error bars, the 95% confidence interval (CI). (B, C) Gene-targeting efficiency in wild-type (C1) and two mRtel1-deficient (A4 and F2) ESC lines at the mRad54 (B) and mPim1 (C) loci. Error bars, the SD from four independent experiments.

FIGURE 5:

mRtel1 is required for telomere replication and extension. (A) Analysis of fragile telomeres in wild-type (C1) and mRtel1-deficient (F2) ESCs with or without 0.2 μM aphidicolin treatment. Bars, SEM; *p < 0.01, **p < 0.001. (B) Sensitivity of wild-type (C1) and two mRtel1-deficient (A4 and F2) ESC lines to the indicated doses of TMPyP4. Bars, mean percentage values of three experiments with SDs. (C) Images from a time-lapse movie of mRtel1-tag-DD and Cherry-mTrf1 coexpressed in mRtel1-deficient (F2) ESCs and cultured in the presence of shield-1 (1 μM). Imaging started 72 h post Cherry-mTrf1 transfection. Ten image z-stacks (0.5-μm spacing) were acquired every 15 min. Far left, three still images (projection of 10 z-stacks) extracted from a time-lapse movie from a single cell acquired at indicated time points. Merge, zooms on the boxed nuclear region of the left images, showing colocalization of mRtel1-tag-DD (third from left) and Cherry-mTrf1 (far right). Scale bar, 5 μm. (D) TRF analysis of wild-type and mRtel1-deficient ESCs. 1, Wild type (C1); 2, mRtel1 deficient (A4); 3, mRtel1 deficient (E1); 4, mRtel1 deficient (F2); 5, wild type (C1 subclone 4); 6, mRtel1 deficient (F2 subclone 11). (E) TRF analysis of wild-type (C1 subclone 4) and mRtel1-deficient (F2 subclones 3 and 11) ESCs. DNA was extracted from wild-type and mRtel1-deficient clones at t = 0 and after 1, 2, and 3 wk in culture.