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

Telomeres shorten with each cell division and can ultimately become substrates for non-homologous end-joining repair, leading to large-scale genomic rearrangements of the kind frequently observed in human cancers. We have characterised over 1400 telomere fusion events at the single-molecule level, using a combination of high-throughput sequence analysis together with experimentally-induced telomeric double-stranded DNA breaks. We show that a single chromosomal dysfunctional telomere can fuse with diverse non-telomeric genomic loci, even in the presence of an otherwise stable genome, and that fusion predominates in coding regions. Fusion frequency was markedly increased in the absence of TP53 checkpoint control and significantly modulated by the cellular capacity for classical, versus alternative, non-homologous end joining (NHEJ). We observed a striking reduction in inter-chromosomal fusion events in cells lacking DNA ligase 4, in contrast to a remarkably consistent profile of intra-chromosomal fusion in the context of multiple genetic knockouts, including DNA ligase 3 and 4 double-knockouts. We reveal distinct mutational signatures associated with classical NHEJ-mediated inter-chromosomal, as opposed to alternative NHEJ-mediated intra-chromosomal telomere fusions and evidence an unanticipated sufficiency of DNA ligase 1 for these intra-chromosomal events. Our findings have implications for mechanisms driving cancer genome evolution

Mechanistic Issues of the Interaction of the Hairpin-Forming Domain of tBid with Mitochondrial Cardiolipin

BACKGROUND: The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated. PRINCIPAL FINDINGS: The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix alphaH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, alphaH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix alphaH6 to efficiently induce cytochrome c release and apoptosis. CONCLUSIONS: Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix alphaH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix alphaH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated

Co-reminiscing with a caregiver about a devastating tornado: Association with adolescent anxiety symptoms

This study explores the association between caregivers’ style of co-reminiscing with their adolescents about an EF4 tornado and youth anxiety symptoms several years following the disaster. Caregiver reward of their children’s emotional expression, defined as attending to and validating emotionally salient content, is generally associated with adaptive youth psychosocial outcomes. However, caregiver reward of youth recollections that are centered around the youth’s negative emotional expression could be an indicator that both caregivers and adolescents are engaged in co-rumination regarding negative emotional experiences. This process may contribute to relatively higher levels of anxiety over time. Adolescents (N = 169) drawn from an ongoing study for aggressive youth (ages 12 to 17; 82% African American) provided individual recollections about their experiences during a devastating tornado 4 to 5 years following the disaster. Caregivers and youth then co-reminisced about their tornado-related experiences. Individual youth recollections were coded for negative personal impact and use of negative emotion words; caregiver–adolescent conversations were coded for caregiver reward of negative emotional expression. Youth who noted more negative personal impacts and used more negative emotion words were higher in parent-rated youth anxiety, and these associations were moderated by caregiver reward of negative emotional expression. The associations between youth recollection qualities and anxiety emerged only when caregivers exhibited high levels of reward of negative emotional expression. These patterns were generally stronger for girls compared to boys. Findings suggest that excessively discussing and rehashing negative experiences, especially several years after the disaster, may be a risk factor for anxiety among disaster-exposed adolescents

POLQ suppresses genome instability and DNA repeat expansions

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ(–)(/–) cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ(–)(/)(–) than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ(–)(/–) cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ(–)(/)(–) cells. In conjunction with the expanded telomere lengths of POLQ(–)(/–) cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease

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

Telomere erosion, dysfunction and fusion can lead to a state of cellular crisis characterized by large-scale genome instability. We investigated the impact of a telomere-driven crisis on the structural integrity of the genome by undertaking whole genome sequence analyses of clonal populations of cells that had escaped crisis. Quantification of large-scale structural variants revealed patterns of rearrangement consistent with chromothripsis, but formed in the absence of functional non-homologous end joining pathways. Rearrangements frequently consisted of short fragments with complex mutational patterns, with a repair topology that deviated from randomness showing preferential repair to local regions or exchange between specific loci. We find evidence of telomere involvement with an enrichment of fold-back inversions demarcating clusters of rearrangements. Our data suggest that chromothriptic rearrangements caused by telomere crisis arise via a replicative repair process involving template switching

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

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

PARP1 is required for preserving telomeric integrity but is dispensable for A-NHEJ

Poly-ADP ribose polymerase 1 (PARP1) is clinically important because of its synthetic lethality with breast cancer allele 1 and 2 mutations, which are causative for inherited breast and ovarian cancers. Biochemically, PARP1 is a single-stranded DNA break repair protein that is needed for preserving genomic integrity. In addition, PARP1 has been implicated in a veritable plethora of additional cellular pathways and thus its precise contribution(s) to human biology has remained obscure. To help address this deficiency, we utilized gene editing to construct genetically-null PARP1 human cancer cells. We found a minor role for PARP1 in an alternative form of DNA double-strand break (DSB) repair, but only when these cells were deficient for the classical form of DSB repair. Despite being proficient for DSB repair, however, cell cycle progression defects and elevated endogenous DNA damage signaling were observed. These deficiencies were instead linked to telomere defects, where PARP1-/- cells had short telomeres that co-localized with markers of endogenous DNA damage and were compromised in their ability to escape a telomere-driven crisis. Our data suggest that while PARP1 does not participate significantly in DNA DSB repair itself, it does prevent the incidence of telomeric DSBs, which, in turn, can drive genomic instability

ATRX modulates the escape from a telomere crisis

Telomerase activity is the principal telomere maintenance mechanism in human cancers, however 15% of cancers utilise a recombination-based mechanism referred to as alternative lengthening of telomeres (ALT) that leads to long and heterogenous telomere length distributions. Loss-of-function mutations in the Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) gene are frequently found in ALT cancers. Here, we demonstrate that the loss of ATRX, coupled with telomere dysfunction during crisis, is sufficient to initiate activation of the ALT pathway and that it confers replicative immortality in human fibroblasts. Additionally, loss of ATRX combined with a telomere-driven crisis in HCT116 epithelial cancer cells led to the initiation of an ALT-like pathway. In these cells, a rapid and precise telomeric elongation and the induction of C-circles was observed; however, this process was transient and the telomeres ultimately continued to erode such that the cells either died or the escape from crisis was associated with telomerase activation. In both of these instances, telomere sequencing revealed that all alleles, irrespective of whether they were elongated, were enriched in variant repeat types, that appeared to be cell-line specific. Thus, our data show that the loss of ATRX combined with telomere dysfunction during crisis induces the ALT pathway in fibroblasts and enables a transient activation of ALT in epithelial cells

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase