DNA Ligase 1 is an essential mediator of sister chromatid telomere fusions in G2 cell cycle phase

Abstract

Fusion of critically short or damaged telomeres is associated with the genomic rearrangements that support malignant transformation. We have demonstrated the fundamental contribution of DNA ligase 4-dependent classical non-homologous end-joining to long-range inter-chromosomal telomere fusions. In contrast, localized genomic recombinations initiated by sister chromatid fusion are predominantly mediated by alternative non-homologous end-joining activity that may employ either DNA ligase 3 or DNA ligase 1. In this study, we sought to discriminate the relative involvement of these ligases in sister chromatid telomere fusion through a precise genetic dissociation of functional activity. We have resolved an essential and non-redundant role for DNA ligase 1 in the fusion of sister chromatids bearing targeted double strand DNA breaks that is entirely uncoupled from its requisite engagement in DNA replication. Importantly, this fusogenic repair occurs in cells fully proficient for non-homologous end-joining and is not compensated by DNA ligases 3 or 4. The dual functions of DNA ligase 1 in replication and non-homologous end-joining uniquely position and capacitate this ligase for DNA repair at stalled replication forks, facilitating mitotic progression.

Tracking telomere fusions through crisis reveals conflict between DNA transcription and the DNA damage response

“…To investigate potential mechanisms for this skew in DNA repair during the progression through crisis, we compared the MH and INS observations from our normal fibroblast dataset with analogous data derived from fibroblasts of patients with DNA ligase 1 and 4 mutations ( Supplementary Figure S3G and H ). In accordance with our previous findings using gene-targeted cancer cell lines ( 27 , 37 ), we discovered a relative decrease in the fraction of genomic compared with intra-chromosomal fusions recovered from the DNA ligase 4-deficient cells with compromised classical NHEJ (CNHEJ) capacity ( Supplementary Figure S3I ). Conversely, fusions derived from DNA ligase 1-deficient fibroblasts were prominently depleted of intra-chromosomal events.…”

“…When charted by frequency ( Supplementary Figure S4E ), intra-chromosomal fusion junctions within the chr17p central subtelomeric region (chr17: 14400–15600) were 1.28-fold ( P = 0.0243) more abundant for the DNA ligase 1-deficient than the normal fibroblasts. Thus, length asymmetry of fused sister chromatids may be coupled with replication, as well as end-joining, mediated by DNA ligase 1 ( 37 ).…”

“…For fibroblast fusion amplicon sequencing, 100 fusion PCR reactions per sample were prepared for quality control and purification of mixed amplicons ahead of Illumina paired-end sequencing. As described previously ( 27 , 37 ), three random PCR wells were selected per sample for amplification verification prior to the 100 replicates for each sample being pooled and purified using Agencourt AMPure XP magnetic beads with elution in nuclease-free water. Aliquots of each sample were taken pre- and post-purification for a comparative assessment and to check purification efficiency.…”

Chromothripsis during telomere crisis is independent of NHEJ, and consistent with a replicative origin

“…Recent studies suggest telomere fusions are mediated by classical (C-NHEJ) and alternative (A-NHEJ) nonhomologous end joining (Smogorzewska et al 2002;Bunting and Nussenzweig 2013;Liddiard et al 2016). C-NHEJ typically involves blunt-end ligation by the XRCC4:LIG4 complex (Critchlow et al 1997;Walker et al 2001;Chang et al 2017), whereas A-NHEJ uses LIG3, with an auxiliary role for LIG1 (Simsek et al 2011;Lu et al 2016;Liddiard et al 2018). A-NHEJ additionally has a requirement for microhomology that can be exposed by preprocessing of the DNA end (Sfeir and Symington 2015; Chang et al 2017).…”

Both the classical and alternative non-homologous end joining pathways contribute to the fusion of drastically shortened telomeres induced by TRF2 overexpression

“…Further experiments are required to examine whether DNA ligase 1 (Lig1) may compensate for the absence of Lig3 and/or Lig4 for TRF2induced chromosome fusions in mechanistically distinct manners. There is precedence for this in at least the case of crisis-induced fusion events [32,33]. Several constituents of the DNA damage repair pathways, such as the Ku complex [5,[34][35][36], WRN [37], PARP1 [38][39][40], and DNA polymerase theta [41], have been reported to affect C-and A-NHEJ choices at damaged telomeres.…”