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Key Points About 10% of all cancers, including some that have a particularly poor prognosis, use the alternative lengthening of telomeres (ALT) pathway to prevent the telomere shortening that accompanies proliferation of normal cells. ALT-positive cells commonly have a number of unusual characteristics, including telomeric DNA that is separated from chromosome ends. This extrachromosomal telomeric DNA may be linear or circular. Partially single-stranded circles of telomeric DNA in which the C-rich (AATCCC)n strand is essentially intact and the G-rich (TTAGGG)n strand is gapped seem to be the best of the known markers for ALT. The quantity of this 'C-circle' DNA correlates well with the amount of ALT activity. Telomere elongation in ALT cells involves homologous recombination. The experimental evidence fits best with a model for ALT in which telomeric 3′ overhangs become extended by invading other telomeric DNA and using it as a template for DNA replication. The other telomeric DNA can be: part of the same telomere (through telomere-loop formation); in a sister chromatid; in the telomere of another chromosome; or in one of the forms of extrachromosomal telomeric DNA. Proteins that are thought to be required for ALT include the homologous recombination protein complexes MRN (which is made up of meiotic recombination 11 (MRE11, also known as MRE11A), RAD50 and Nijmegen breakage syndrome 1 (NBS1, also known as NBN)) and structural maintenance of chromosomes 5 (SMC5)–SMC6, and proteins, such as flap endonuclease 1 (FEN1), MUS81, Fanconi anaemia group D2 (FANCD2) and Fanconi anaemia group A (FANCA), that may be required for recombination-dependent restart of stalled telomeric DNA replication. Promyelocytic leukaemia (PML) bodies containing telomeric DNA are characteristic of ALT cells, and are referred to as ALT-associated PML bodies (APBs). Large APBs seem to be associated with senescence of ALT cells and sequestration of extrachromosomal DNA, but we speculate that smaller APBs may be sites at which telomere lengthening occurs. In ALT cells, many of the telomeres elicit a DNA-damage response but repress chromosome end-to-end fusions. This telomere state, which is intermediate between the fully capped and uncapped fusogenic telomere states, may reflect a structural change that is permissive for recombination-mediated telomere replication. A hallmark of cancer cells is their ability to prevent telomere shortening. Sometimes this is achieved without telomerase by a process known as alternative lengthening of telomeres (ALT). Recent progress has been made in understanding how ALT occurs. Unlimited cellular proliferation depends on counteracting the telomere attrition that accompanies DNA replication. In human cancers this usually occurs through upregulation of telomerase activity, but in 10–15% of cancers — including some with particularly poor outcome — it is achieved through a mechanism known as alternative lengthening of telomeres (ALT). ALT, which is dependent on homologous recombination, is therefore an important target for cancer therapy. Although dissection of the mechanism or mechanisms of ALT has been challenging, recent advances have led to the identification of several genes that are required for ALT and the elucidation of the biological significance of some phenotypic markers of ALT. This has enabled development of a rapid assay of ALT activity levels and the construction of molecular models of ALT.

Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.Lamm et al. report that replication stress activates mTOR through ATR to induce nuclear actin polymerization, facilitating the recovery from replication stress.

Cells that bypass senescence in the absence of the p53 tumour suppressor protein have shortened telomeres that undergo fusion, and these fusions trigger mitotic arrest and cell death in crisis. DNA damage signal kills cancer cells in crisis Cells forming a tumour must overcome two barriers before becoming cancerous. The first is senescence and the second is a proliferative block known as crisis. Cells that escape senescence usually succumb during crisis, but it has not been clear what triggers cell death at that stage. Jan Karlseder and colleagues now demonstrate that cells that bypass senescence in the absence of p53 have shortened telomeres that undergo fusion, and these fusions trigger mitotic delay. During mitotic arrest telomeres are further deprotected and detected by the DNA damage machinery, which leads to cell death. These findings might offer a clinical opportunity, as exacerbation of mitotic telomere deprotection sensitizes cancer cells to mitotic drugs — but mitotic arrest has also been associated with genome instability and tumorigenesis in checkpoint-compromised cells. Tumour formation is blocked by two barriers: replicative senescence and crisis 1 . Senescence is triggered by short telomeres and is bypassed by disruption of tumour-suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbour unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remains unexplained. Here we show that human cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. This phenotype is induced by loss of p53 function, and is suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating that such fusions are the underlying cause of cell death. Exacerbation of mitotic telomere deprotection by partial TRF2 (also known as TERF2 ) knockdown 2 increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population.

To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia–telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.

Replicative senescence is accompanied by a telomere‐specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early‐passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end‐to‐end fusions and mostly result from strand‐independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1‐phase cells just before senescence and after bypassing senescence by inactivation of wild‐type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53‐dependent senescence. Replicative senescence is triggered by DNA damage response foci associated with telomeres. Reddel and colleagues now establish that a threshold of five damaged telomeres exists to induce senescence in normal cells and that end‐to‐end chromosome fusion is not required for senescence induction.

The recruitment of telomerase to telomeres is a tightly regulated process which is stimulated by replication stress and the DNA damage response regulatory kinase ATR, via an unknown mechanism. Here, we demonstrate that nuclear filamentous actin is important for the stable interaction of telomerase with telomeres in immortal human cells, resulting in productive telomere elongation by telomerase in an actin-dependent manner. This process is regulated by both ATR and mTOR kinases, and employs other regulators of actin structure and function, such as WASP, ARP2/3 and myosin. Nuclear filamentous actin serves as a site for telomerase recruitment, which is mediated by telomere tethering on actin fibers in response to replication stress, allowing telomerase to localize to telomeres containing stalled replication forks. Overall, these data demonstrate that, in human cells which express telomerase, telomeric replication stress triggers the recruitment of telomerase to telomeres via a nuclear actin network, enabling telomere length maintenance. Telomerase recruitment to telomeres is a tightly regulated process which is stimulated by replication stress. Here, the authors identify that nuclear filamentous actin is important for interaction between telomerase and telomeres, ultimately facilitating productive telomere extension by telomerase.

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway. Here, the authors characterise the zinc finger protein ZNF827 as a single stranded DNA binding protein that accumulates at stalled replication forks to activate the ATR-CHK1 pathway and engage homologous-recombination mediated DNA repair.

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis. The Eyes Absent proteins (EYA1-4) are a group of tyrosine phosphatases. Here, the authors report a signalling pathway in which EYA4 and EYA1 dephosphorylate Polo-like kinase 1 (PLK1) at pY445 to support PLK1 activation and mitosis.

Telomeric DNA is protected by the shelterin complex, whose disruption triggers DNA-damage responses, checkpoint activation and chromosomal fusions. Now analysis of human cell lines reveals a spontaneously occurring intermediate state in which the DNA-damage response is activated at the telomeres without cell cycle arrest or chromosomal fusions, and with TRF2 playing a central role in determining such a state. Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR + telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity–positive (TA + ) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT + ), DDR is partly independent of telomere length, mostly affects leading strand–replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT + (but not TA + ) cells, DDR + telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR + telomeres therefore arise through different mechanisms in TA + and ALT + cells and have different consequences.

Telomeres prevent ATM activation by sequestering chromosome termini within telomere loops (t-loops). Mitotic arrest promotes telomere linearity and a localized ATM-dependent telomere DNA damage response (DDR) through an unknown mechanism. Using unbiased interactomics, biochemical screening, molecular biology, and super-resolution imaging, we found that mitotic arrest-dependent (MAD) telomere deprotection requires the combined activities of the Chromosome passenger complex (CPC) on shelterin, and the BLM-TOP3A-RMI1/2 (BTR) complex on t-loops. During mitotic arrest, the CPC component Aurora Kinase B (AURKB) phosphorylated both the TRF1 hinge and TRF2 basic domains. Phosphorylation of the TRF1 hinge domain enhances CPC and TRF1 interaction through the CPC Survivin subunit. Meanwhile, phosphorylation of the TRF2 basic domain promotes telomere linearity, activates a telomere DDR dependent on BTR-mediated double Holliday junction dissolution, and leads to mitotic death. We identify that the TRF2 basic domain functions in mitosis-specific telomere protection and reveal a regulatory role for TRF1 in controlling a physiological ATM-dependent telomere DDR. The data demonstrate that MAD telomere deprotection is a sophisticated active mechanism that exposes telomere ends to signal mitotic stress. Here the authors reveal how telomeres signal mitotic stress. A key protein network alters their structure exposing telomere ends to signal mitotic stress, ultimately triggering a controlled DNA damage response to remove faulty cells.