Escape from Telomere-Driven Crisis Is DNA Ligase III Dependent

Abstract

Short dysfunctional telomeres are capable of fusion, generating dicentric chromosomes and initiating breakage-fusion-bridge cycles. Cells that escape the ensuing cellular crisis exhibit large-scale genomic rearrangements that drive clonal evolution and malignant progression. We demonstrate that there is an absolute requirement for fully functional DNA ligase III (LIG3), but not ligase IV (LIG4), to facilitate the escape from a telomere-driven crisis. LIG3- and LIG4-dependent alternative (A) and classical (C) nonhomologous end-joining (NHEJ) pathways were capable of mediating the fusion of short dysfunctional telomeres, both displaying characteristic patterns of microhomology and deletion. Cells that failed to escape crisis exhibited increased proportions of C-NHEJ-mediated interchromosomal fusions, whereas those that escaped displayed increased proportions of intrachromosomal fusions. We propose that the balance between inter- and intrachromosomal telomere fusions dictates the ability of human cells to escape crisis and is influenced by the relative activities of A- and C-NHEJ at short dysfunctional telomeres.

Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4

“…LIG3-mediated ligation is associated with microhomology usage, whereas the rapid kinetics of LIG4-mediated C-NHEJ generally preclude extensive processing and resection required for homology searching (Shibata et al 2011). Consistent with our published observations (Letsolo et al 2010;Jones et al 2014), we found that mean microhomology usage at inter-chromosomal junctions was greater for LIG4 −/− cells (2.38 nt) than LIG3 −/− cells (1.44 nt) ( Fig. 5A; Supplemental Fig.…”

“…Physiologically, all lines experienced a 65%-83% reduction in cell number 48 h (t48) post-nucleofection (Supplemental Fig. 2E), consistent with an episode of mitotic arrest and telomere fusion akin to a telomeric crisis-state (Jones et al 2014). The WT populations recovered most rapidly, with a 10-fold increase in cell number between 48 and 120 h (t120), coincident with a threefold reduction in 17p fusion frequency (Fig.…”

“…Although no definitive inter-sample variation in ontological associations is present, the enrichment of genes involved in cell division and chromosome partitioning disrupted in LIG4 −/− and LIG3 −/−:NC3 samples with potentially enhanced A-NHEJ function remained statistically significant after multiple testing correction (P = 0.017). This is intriguing given the propensity of these cells for escape from telomere-driven crisis and cellular transformation via telomerase reactivation (Jones et al 2014). Conjointly, we sequence-verified fusion junctions within the HMGA2 (Li et al 2011) gene (Chr12q14.3) and Chr3p21.31 region implicated in the regulation of telomerase activity ( Fig.…”

“…We have proposed that the capacity of cells to escape from a telomere-driven crisis may depend on the relative balance between short-range localized intra-chromosomal recombination and more extensive LIG4-dependent inter-chromosomal interactions incompatible with proper chromosomal segregation and mitosis (Jones et al 2014). We considered that different processes might operate to catalyze inter-compared with intra-chromosomal fusions.…”

“…The interdependence or hierarchy of specialized LIG1 and LIG3 roles in A-NHEJ remains to be clarified in human cells. We have previously documented the essential role of LIG3 in the fusion of critically short telomeres capping sister chromatids in cells undergoing a telomere-driven crisis (Jones et al 2014). We consider that such intra-chromosomal telomere fusions may endow cells with the capacity for escape and recovery of mitotic function following localized sequence amplifications or deletions (Murnane 2010) that ultimately up-regulate telomerase.…”