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.Mammalian telomeres solve the end protection problem through their association with shelterin. The shelterin factor TRF2 (telomere repeat-binding factor 2) protects chromosome ends from inappropriate DNA repair events that threaten the integrity of the genome (reviewed in reference 32). When TRF2 is removed by Cre-mediated deletion from conditional knockout mouse embryo fibroblasts (TRF2 F/Ϫ MEFs), telomeres activate the ATM kinase pathway and are processed by the canonical nonhomologous end-joining (NHEJ) pathway to generate chromosome end-to-end fusions (10, 11).The repair of telomeres in TRF2-deficient cells is readily monitored in metaphase spreads. Over the course of four or five cell divisions, the majority of chromosome ends become fused, resulting in metaphase spreads displaying the typical pattern of long trains of joined chromosomes (10). The reproducible pace and the efficiency of telomere NHEJ have allowed the study of factors involved in its execution and regulation. In addition to depending on the NHEJ factors Ku70 and DNA ligase IV (10, 11), telomere fusions are facilitated by the ATM kinase (26). This aspect of telomere NHEJ is mediated through the ATM kinase target 53BP1. 53BP1 accumulates at telomeres in TRF2-depleted cells and stimulates chromatin mobility, thereby promoting the juxtaposition of distantly positioned chromosome ends prior to th...

Shelterin complex and associated factors at human telomeres

“…44,81,82 The data is compatible with two roles. First, the MRN complex mediates, at least in part, the ATM response leading to TIF formation after TRF2 deletion.…”

“…As a result, the amounts of telomere fusions are highly reduced (from 40% fusions per chromosome, to 15%). 82 The same conclusions were drawn from the analysis of NBS1 -/-, 81 or MRE11 -/-, 44 cells. Second, the MRN complex affects the processing of damaged telomeres by influencing the production of the overhang from a blunt end telomere, obtained after telomere replication.…”

Single strand DNA binding proteins 1 and 2 protect newly replicated telomeres

“…Chk2 phosphorylation was markedly decreased in shTRF2-treated mSSB1 ∆/∆ ; mS-SB1 Y85A MEFs ( Figure 4A). As end-to-end chromosome fusions following TRF2 depletion depends strictly on an intact ATM-Chk2 pathway [23,24], this result suggests that like hSSB1, both mSSB1 and mSSB2 activate the ATM-Chk2 pathway following DNA damage [16][17][18]20]. Our results contrast with a recent report documenting that mSSB1 and mSSB2 do not play roles in ATMdependent DDR in primary lymphocytes and MEFs [27].…”

“…Telomeres depleted of TRF2 only activate the ATMChk2-dependent DNA damage signaling pathway and are repaired by classical NHEJ (C-NHEJ) [23,24]. We therefore monitored the number of chromosome fusions observed in TRF2-deficient WT and mSSB1 ∆/∆ MEFs.…”

PHF11 promotes DSB resection, ATR signaling, and HR

“…The accumulation of PHF11 at some of the telomeres lacking TRF2 is consistent with the identification of PHF11 in the PICh experiment. In the absence of TRF2, telomeres undergo limited ATM/CtIP-dependent resection but do not activate ATR and are not likely to contain RPA, since the telomeres retain the POT1 proteins, which exclude RPA from the single-stranded telomeric DNA (Denchi and de Lange 2007;Dimitrova and de Lange 2009;Gong and de Lange 2010;Lottersberger et al 2013). Thus, at some of the telomeres lacking TRF2, the PHF11 foci are detectable despite the presumed absence of RPA and ATR signaling.…”