Cell cycle-dependent role of MRN at dysfunctional telomeres: ATM signaling-dependent induction of nonhomologous end joining (NHEJ) in G1 and resection-mediated inhibition of NHEJ in G2

Abstract

Here, we address the role of the MRN (Mre11/Rad50/Nbs1) complex in the response to telomeres rendered dysfunctional by deletion of the shelterin component TRF2. Using conditional NBS1/TRF2 double-knockout MEFs, we show that MRN is required for ATM signaling in response to telomere dysfunction. This establishes that MRN is the only sensor for the ATM kinase and suggests that TRF2 might block ATM signaling by interfering with MRN binding to the telomere terminus, possibly by sequestering the telomere end in the t-loop structure. We also examined the role of the MRN/ATM pathway in nonhomologous end joining (NHEJ) of damaged telomeres. NBS1 deficiency abrogated the telomere fusions that occur in G(1), consistent with the requirement for ATM and its target 53BP1 in this setting. Interestingly, NBS1 and ATM, but not H2AX, repressed NHEJ at dysfunctional telomeres in G(2), specifically at telomeres generated by leading-strand DNA synthesis. Leading-strand telomere ends were not prone to fuse in the absence of either TRF2 or MRN/ATM, indicating redundancy in their protection. We propose that MRN represses NHEJ by promoting the generation of a 3' overhang after completion of leading-strand DNA synthesis. TRF2 may ensure overhang formation by recruiting MRN (and other nucleases) to newly generated telomere ends. The activation of the MRN/ATM pathway by the dysfunctional telomeres is proposed to induce resection that protects the leading-strand ends from NHEJ when TRF2 is absent. Thus, the role of MRN at dysfunctional telomeres is multifaceted, involving both repression of NHEJ in G(2) through end resection and induction of NHEJ in G(1) through ATM-dependent signaling.

FIG. 1.

The MRN complex is the sensor in the ATM pathway. (A) Immunoblots detecting NBS1, TRF2, ATM-S1981-P, and Chk2 phosphorylation in indicated MEFs, harvested untreated or 72 h after Cre infection. γ-Tubulin was used as a loading control. * indicates a nonspecific band. (B) Effects of NBS1 deletion on γ-H2AX, MDC1, and 53BP1 TIF formation. TRF2^F/F NBS1^F/+ and TRF2^F/F NBS1^F/− MEFs, harvested 72 h after Cre treatment and processed for immunofluorescence-FISH (γ-H2AX, MDC1, or 53BP1 [green] costained with telomeric TTAGGG-specific FISH probe [red] or DAPI [blue]). Images were merged and enlarged. (C) Frequency of TIF-positive cells. At least 150 cells processed as described for panel B were scored for 10 or more telomeric 53BP1 foci. Average values and standard deviations for three independent experiments are indicated.

FIG. 2.

Effect of NBS1 deficiency on telomere fusions. (A) Metaphase spreads from TRF2^F/F NBS1^F/+ and TRF2^F/F NBS1^F/− MEFs, harvested untreated or 96 and 120 h after Cre infection. Telomeric signals were detected with FITC-OO-(CCCTAA)₃ oligonucleotide (green), and DNA was stained with DAPI (red). Enlarged panels show representative chromosome- and chromatid-type fusions. (B) Schematic of the method used to determine the frequency of fused ends in metaphase spreads containing chromosome-type, chromatid-type, and sister telomere fusion events. (C) Quantification of the frequency of chromosome ends engaged in chromosome- and chromatid-type fusions, scored as described for panel B, in metaphase spreads of cells shown in panel A. Bars represent averages of results from three independent experiments, and error bars indicate standard deviations. At least 2,000 chromosome ends were scored for each cell line and treatment. (D) Quantification of the frequency of chromosome ends engaged in chromatid-type and sister telomere fusions, scored as described for panel B, in metaphase spreads of TRF2^F/F NBS1^F/+ and TRF2^F/F NBS1^F/− MEFs, harvested at 96 h after Cre treatment, in the presence or absence of 50 μM roscovitine for 4 h prior to harvesting. Error bars represent standard deviations from the average values for three independent experiments. At least 2,000 chromosome ends were scored for each cell line and treatment.

FIG. 3.

NBS1 prevents the fusions of telomeres generated by leading-strand DNA synthesis. (A) Schematic of the possible telomere fusion events that can be detected in metaphase spreads. (Top) The fusion of dysfunctional telomeres before replication (G₀ or G₁) leads to the occurrence of chromosome-type fusions in metaphase spreads. (Bottom) Postreplicative (S or G₂) repair of dysfunctional telomeres can give rise to sister, chromosome-type, or chromatid-type telomere fusions. The different combinations of joining events between leading- and lagging-strand chromatids are indicated. (B) Schematic of the CO-FISH method used to differentiate telomeres resulting from leading- and lagging-strand DNA synthesis. (C) Metaphase spreads of TRF2^F/F NBS1^F/+, TRF2^F/F NBS1^F/−, and TRF2^+/+ NBS1^F/− MEFs, analyzed by CO-FISH at 120 h after Cre treatment. Telomeric signals on the leading strand were detected with TAMRA-OO-(TTAGGG)₃ oligonucleotide (red) and costained with FITC-OO-(CCCTAA)₃ probe (green) specific to lagging-strand telomeres; DNA was stained with DAPI (blue). Enlarged panels show representative fusion events. (D) Table showing the chromatid-type fusions detected in metaphase spreads of Cre-treated TRF2^F/F NBS1^F/+, TRF2^F/F NBS1^F/−, and TRF2^+/+ NBS1^F/− cells (shown in panel C). The total number of chromosome ends, the number of chromatid ends fused, and the number of leading ends fused to leading ends are indicated. The data were confirmed in an independent experiment. At least 3,000 chromosome ends were scored for each cell line.

FIG. 4.

Telomeric overhangs in TRF2- and NBS1-deficient cells. (A) Telomeric DNA analysis. (Left) In-gel assay detecting 3′ overhang of TRF2^F/F NBS1^F/+ and TRF2^F/F NBS1^F/− MEFs, harvested untreated or 96 and 120 h after infection with Cre and processed by in-gel hybridization to a (CCCTAA)₄ probe to detect single-stranded TTAGGG repeats (native). (Right) The DNA was denatured in situ and rehybridized to the same probe to detect the total TTAGGG signal (denatured). Overhang signals were quantified with ImageQuant software and normalized to the total TTAGGG signal in the same lane. The numbers below the gel represent the percentages of normalized overhang signal compared to the normalized overhang signal for the same cells not treated with Cre. (B) Quantification of telomeric overhang at 0, 96, and 120 h after infection with Cre in five independent experiments. The overhang signals at different time points after Cre infection are represented as percentages of the overhang signal in the absence of Cre for the same cell line.

FIG. 5.

ATM protects leading-strand telomeres from processing by NHEJ upon loss of TRF2. (A) Immunoblot detection of ATM, TRF2, and Chk2 in TRF2^F/− ATM^+/−, TRF2^F/− ATM^−/−, and TRF2^F/− ATM^−/− Lig4^−/− MEFs untreated or 72 h after Cre infection. γ-Tubulin was used as a loading control. * indicates a nonspecific band. (B) Metaphase spreads of TRF2^F/− ATM^+/−, TRF2^F/− ATM^−/−, and TRF2^F/− ATM^−/− Lig4^−/− MEFs, analyzed by CO-FISH untreated or 120 h after Cre treatment. Telomeric signals on the leading strand were detected with TAMRA-OO-(TTAGGG)₃ oligonucleotide (red) and costained with a lagging-strand telomere-specific FITC-OO-(CCCTAA,)₃ probe (green); DNA was stained with DAPI (blue). (C) Quantification of chromosome- and chromatid-type fusions detected in metaphase spreads of cells shown in panel B. (D) Table showing the chromatid-type fusions detected in metaphase spreads shown in panel B and scored in panel C. The total number of chromosome ends, the number of chromatid ends fused, and the number of leading ends fused to leading ends are indicated. At least 2,000 chromosome ends were scored for each cell line.

FIG. 6.

Model for the dual role of MRN/ATM at functional and dysfunctional telomeres. (A) At functional telomeres, prior to replication, TRF2-mediated t-loop formation or maintenance is proposed to block the loading of Ku70/80 and MRN onto telomeric DNA, thus protecting chromosome ends from processing by NHEJ and from activating ATM signaling. (B) Model for repression of NHEJ at telomeres replicated by leading-strand DNA synthesis. After replication, TRF2 is proposed to promote overhang generation by recruiting MRN and an unknown nuclease(s), both of which have the ability to resect blunt telomeres generated by leading-strand DNA synthesis. The overhang may block NHEJ through its interaction with POT1. In the absence of TRF2, MRN/ATM-dependent DSB processing of dysfunctional telomeres also generates resected ends that can bind POT1 and are protected from NHEJ.