Multiple roles for MRE11 at uncapped telomeres

Abstract

Progressive telomere attrition or uncapping of the shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. Telomere deprotection activates both ataxia telangiectasia mutated (ATM) and telangiectasia and Rad3-related (ATR) kinase-dependent DNA damage response pathways, and promotes efficient non-homologous end-joining (NHEJ) of dysfunctional telomeres. The mammalian MRE11-RAD50-NBS1 (MRN; NBS1 is also known as NBN) complex interacts with ATM to sense chromosomal double-strand breaks and coordinate global DNA damage responses. Although the MRN complex accumulates at dysfunctional telomeres, it is not known whether mammalian MRN promotes repair at these sites. Here we address this question by using mouse alleles that either inactivate the entire MRN complex or eliminate only the nuclease activities of MRE11 (ref. 8). We show that cells lacking MRN do not activate ATM when telomeric repeat binding factor 2 (TRF2) is removed from telomeres, and ligase 4 (LIG4)-dependent chromosome end-to-end fusions are markedly reduced. Residual chromatid fusions involve only telomeres generated by leading strand synthesis. Notably, although cells deficient for MRE11 nuclease activity efficiently activate ATM and recruit 53BP1 (also known as TP53BP1) to deprotected telomeres, the 3' telomeric overhang persists to prevent NHEJ-mediated chromosomal fusions. Removal of shelterin proteins that protect the 3' overhang in the setting of MRE11 nuclease deficiency restores LIG4-dependent chromosome fusions. Our data indicate a critical role for the MRN complex in sensing dysfunctional telomeres, and show that in the absence of TRF2, MRE11 nuclease activity removes the 3' telomeric overhang to promote chromosome fusions. MRE11 can also protect newly replicated leading strand telomeres from NHEJ by promoting 5' strand resection to generate POT1a-TPP1-bound 3' overhangs.

Figure 1. Activation of ATM, not ATR, following TRF2 depletion requires the MRN complex but not Mre11 nuclease activity

a, b, γ-H2AX-positive TIFs in MEFs of the indicated genotypes after TRF2 depletion in Mre11 deficient SV40LT immortalized MEFs (a) and Mre11 nuclease activity deficient MEFs (b). c, d, Western blots detecting TRF2, Mre11, ATM and Chk2 phosphorylation after shTRF2 treatment in Mre11 deficient SV40LT immortalized MEFs (c) and Mre11 nuclease activity deficient MEFs (d). Tubulin served as loading control. e, f, Immunoblots for Mre11 and ATR phosphorylation and RT-PCR to detect Pot1a and GAPDH transcripts after shPot1a infection of Mre11 deficient SV40LT immortalized MEFs (e) and Mre11 nuclease activity deficient MEFs (f). Nsp: non-specific protein served as loading control.

Figure 2. The MRN complex and Mre11 nuclease activity are required for NHEJ of telomeres lacking TRF2

a, MEFs of the indicated genotypes were treated with control vector or shTRF2 for 120h, metaphases were prepared and telomere fusions were visualized by telomere PNA-FISH (red) and DAPI (blue). b, In-gel hybridization assay using a CHEF gel to fractionate genomic DNA, then hybridized in situ to a (CCCTAA)₄ probe to detect the 3’ overhang under native conditions (left) and under denatured condition (right) to detect total TTAGGG repeats (right). c, (Left panel): Telomeric probes used in CO-FISH experiments [FITC-OO-(TTAGGG)₄; green, labels the leading strand and Tam-OO-(CCCTAA)₄; red, labels the lagging strand] and schematic of fusion products expected in chromosome-chromosome, leading-leading chromatid and lagging-lagging chromatid fusions. (Right panel): Fusion products of shTRF2 treated cells of the indicated genotypes analyzed by CO-FISH. DNA was detected by DAPI (blue). Representative images are shown. d, Quantification of the types of fusions observed in cells of the indicated genotypes as a percentage of total fusions observed following TRF2 depletion. A minimum of 150 independent fusion events with telomeric signals at the sites of fusions were characterized per genotype.

Figure 3. Pot1a-TPP1 complex protects single-stranded overhangs from engaging in inappropriate repair

a, Depletion of Pot1a or TPP1, but not Pot1b, results in pronounced telomere fusions in metaphase spreads from shTRF2 treated Mre11^H129N/Δ MEFs, with telomeric PNA-FISH (red) and DAPI (blue). b, Quantitation of telomere fusions from representative images are shown in (b). Error bars, s.d.; n ≥ 350, asterisk, p<0.001. c, Depletion of ATR or 53BP1 inhibits chromosomal fusions generated by expression of TPP1^ΔRD cDNA and removal of TRF2 in Mre11^H129N/Δ MEFs. d, Qunatification of telomere fusions from representative images shown in (c). Error bars, s.d.; n ≥ 900, asterisk, p<0.001.

Figure 4. Ligase IV is required for NHEJ of single-stranded telomeric overhangs

a, Immunoblot demonstrating that shLig4 efficiently depletes Lig4 from Mre11^H129N/Δ nuclease-deficient MEFs expressing TPP1^ΔRD and shTRF2. Nsp: nonspecific protein used as loading control. b, Lig4 is required for chromosomal fusions in Mre11^H129N/Δ nuclease-deficient MEFs expressing TPP1^ΔRD and shTRF2. c, Quantitation of number of fusions per chromosome from representative images shown in (b). Error bars, s.d.; n ≥ 850, asterisk, p<0.001. d, T-SCEs in Mre11^H129N/Δ nuclease-deficient MEFs deficient in Lig4, TRF2 and TPP1. CO-FISH was performed using FITC-OO-TTAGGG₄ (green) and Tam-OO-(CCCTAA)₄ (red) probes.