The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres

Abstract

Repair of DNA double‐stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non‐homologous end‐joining (NHEJ) or error‐free homologous recombination. NHEJ precedes either by a classic, Lig4‐dependent process (C‐NHEJ) or an alternative, Lig4‐independent one (A‐NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere‐binding proteins are recognized as DSBs. In this report, we studied which end‐joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end‐to‐end chromosome fusions mediated by the C‐NHEJ pathway. In contrast, removal of Tpp1–Pot1a/b initiated robust chromosome fusions that are mediated by A‐NHEJ. C‐NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A‐NHEJ is a major pathway to process dysfunctional telomeres.

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].…”

“…Consistent with an overall decrease in damage signaling at dysfunctional telomeres (Supplementary information, Figure S3), Chk1 phosphorylation was reduced by mSSB1 deletion in MEFs treated with shTRF2 or TPP1 ∆RD ( Figure 2G). Taken together, our results suggest that mSSB1 promotes C-NHEJ-mediated DNA repair while repressing alternative-NHEJ (A-NHEJ) mediated repair at dysfunctional telomeres, as chromosome fusions in cells devoid of TPP1-POT1a/b arise from A-NHEJ-mediated repair [24].…”

“…mSSB1 ∆/∆ MEFs show a significant decrease in the number of end-to-end chromosome fusions after removal of TRF2, due to decreased activation of the ATM-Chk2 signaling pathway essential for C-NHEJ-mediated repair of telomeres [24,28]. In addition, mSSB1 and mSSB2 function to protect newly replicated Goverhangs.…”

“…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.…”

The mINO80 chromatin remodeling complex is required for efficient telomere replication and maintenance of genome stability

“…We rendered telomeres dysfunctional in CAG-CreER; mIno80 F/F or CAGCreER; mIno80 ∆/∆ MEFs by either removing endogenous mPOT1a and mPOT1b proteins from telomeres using a dominant negative TPP1 mutant (TPP1 ∆RD ), or by shRNA-mediated depletion of endogenous TRF2 [13,16,43,44]. mIno80 deletion did not impact upon telomere dysfunction-induced DNA damage focus (TIF) formation ( Figure 5A and 5B and Supplementary information, Figure 4C and 4D), consistent with our finding that mIno80 deletion did not disrupt IR-induced DNA damage focus formation on genomic DNA (Figure 3).…”