Stop pulling my strings - what telomeres taught us about the DNA damage response

Abstract

Mammalian cells have evolved specialized mechanisms to sense and repair double-strand breaks (DSBs) to maintain genomic stability. However, in certain cases, the activity of these pathways can lead to aberrant DNA repair, genomic instability and tumorigenesis. One such case is DNA repair at the natural ends of linear chromosomes, known as telomeres, which can lead to chromosome-end fusions. Here, we review data obtained over the past decade and discuss the mechanisms that protect mammalian chromosome ends from the DNA damage response. We also discuss how telomere research has helped to uncover key steps in DSB repair. Last, we summarize how dysfunctional telomeres and the ensuing genomic instability drive the progression of cancer.

Figure 1. Overview of telomere composition and function

Mammalian telomeres are composed of long stretches of TTAGGG repeats that range from 5 kb in human cells to 100 kb in mice and end with a single-stranded 3′ overhang of up to a few hundred nucleotides in length^,. Telomeric DNA is bound by the specialized shelterin complex, transcribed into a long non-coding telomeric repeat-containing RNA (TERRA) and packaged into a t-loop (telomere loop) configuration. Shelterin subunits include TRF1 (telomere repeat-binding factor 1), TRF2, TIN2 (TRF1-interaction factor 2), RAP1 (repressor activator protein 1), TPP1 and POT1 (protection of telomere 1; POT1A and POT1B in mice). The six-subunit complex protects chromosome ends from DNA damage signalling by ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related), and from DNA repair by c-NHEJ (classical non-homologous end joining), alt-NHEJ (alternative non-homologous end joining), HR (homologous recombination) and DNA end resection.

Figure 2. How shelterin protects telomeres

a | TRF2 (telomere repeat-binding factor 2) represses ATM (ataxia telangiectasia mutated) signalling and classical non-homologous end joining (c-NHEJ). TRF2 promotes the formation of the protective telomere loop (t-loop) structure, which hides chromosome ends from ATM and c-NHEJ. In addition, TRF2 inhibits 53BP1 (p53-binding protein 1) accumulation by blocking RNF168-mediated ubiquitylation by activating the deubiquitylase BRCC3 (BRCA1/BRCA2-containing complex, subunit 3). Last, TRF2 blocks the dimerization of the Ku complex, thereby preventing the activation of c-NHEJ. b | POT1 (protection of telomere 1) represses ATR (ataxia telangiectasia and Rad3-related) signalling by competing with RPA (replication protein A) for single-stranded DNA (ssDNA) binding at telomeres. c | TRF1 inhibits ATR activity during telomere replication with the help of TPP1–POT1. TRF1 also counteracts replication fork stalling at telomeric secondary DNA structures (such as quadruplex DNA (G4)) with the help of RTEL1 (regulator of telomere elongation helicase 1) and BLM (Bloom syndrome, RecQ helicase-like), thereby protecting against telomere fragility. RTEL1 is recruited to replicating telomeres by interacting with PCNA (proliferating cell nuclear antigen). d | Alt-NHEJ (alternative-NHEJ), which is dependent on DNA ligase 3 (LIG3), PARP1 (poly(ADP-ribose) polymerase 1) and DNA polymerase θ (Pol θ), is repressed in a redundant manner by shelterin and the Ku70–Ku80 complex. e | The generation of telomere 3′ overhang involves TRF2-dependent recruitment of the nuclease Apollo to resect double-stranded ends. Leading and lagging ends are then resected by EXO1 (exonuclease 1) to generate long single-stranded overhangs, which are subsequently filled in by Polα–primase and the CST (CTC1–STN1–TEN1) complex. f | Aberrant resection of uncapped telomeres is carried out by the enzymatic machinery that processes double-strand breaks (DSBs)—the nucleases CtIP (CtBP-interacting protein) and EXO1 and the helicase BLM — and is repressed redundantly by shelterin and 53BP1. iDDR, inhibitor of the DNA damage response.

Figure 3. The three facets of telomere homologous recombination: T-SCE (telomere sister chromatid exchange), t-loop (telomere loop) homologous recombination and ALT (alternative lengthening of telomeres)

a | Exchange of sequence between sister chromatid telomeres (marked in red and green) is inhibited by RAP1 (repressor activator protein 1), POT1 (protection of telomere 1) and Ku70–Ku80. b | T-loop homologous recombination is blocked by TRF2 (telomere repeat-binding factor 2). TRF2 recruits RTEL1 (regulator of telomere elongation helicase 1) during S phase to unwind the t-loop and therefore protect it from being cleaved by structure-specific endonuclease subunit SLX4. In addition, TRF2 inhibits t-loop excision by inhibiting the activity of NBS1–XRCC3 (Nijmegen breakage syndrome 1–X-ray repair complementing defective repair in Chinese hamster cells 3). c | Telomere repeats have the propensity to form stable quadruplex (G4) DNA structures, which would impede replication fork progression. It has been proposed that ATRX (α-thalassaemia/mental retardation syndrome X-linked) unwind G4 DNA, enabling the deposition of histone H3.3 and ultimately assisting replication fork progression. The activity of ATRX at telomeres inhibits various ALT (alternative lengthening of telomeres) phenotypes including T-SCEs, formation of telomere circles, intrachromosomal telomere recombination and formation of APBs (ALT-associated promyelocytic leukaemia nuclear bodies). DAXX, death domain-associated protein; HR, homologous recombination; PML, promyelocytic leukaemia; RPA, replication protein A.

Figure 4. Telomeres as a tool to investigate DNA end resection and classical non-homologous end joining (c-NHEJ)

a | An overview of the assay to monitor c-NHEJ and DNA end resection at telomeres. Southern blot analysis allows the visualization of telomere fusion events. Genomic DNA is cleaved with frequently cutting restriction enzymes, resolved on a denaturing gel and hybridized with a radiolabelled telomere probe. As TTAGGG repeats are not cut by restriction enzymes, they are resolved according to their length (in range of the solid vertical line). Telomere fusions that occur following telomere repeat-binding factor 2 (TRF2) depletion are delineated as slow-migrating restriction fragments (dotted vertical line). Inhibition of factors that promote end joining, one example being p53-binding protein 1 (53BP1), prevents the accumulation of these long restriction fragments. This assay can be adjusted to quantify the length of the 3′ overhang, which is generated by 5′ end resection. Specifically, in-gel hybridization is carried out using a radiolabelled telomere probe in native conditions, in which the probe only hybridizes to the terminal single-stranded DNA (ssDNA) telomere overhang. The overhang signal in the native gel is quantified and normalized to the total telomeric DNA. Depletion of factors, including 53BP1 and RAP1-interacting factor 1 (RIF1), that block end resection will lead to excess overhang signal and can be readily examined with this assay. b | A schematic representing key players that promote c-NHEJ and block DNA end resection, focusing on factors that were studied in the context of dysfunctional telomeres in TRF2-deficient cells. Double-strand break (DSB) sensing by the MRE11–RAD50–NBS1 (MRN)^– complex triggers a signalling cascade by recruiting autophosphorylated ataxia telangiectasia mutated (ATM). ATM then phosphorylates the histone variant H2A.X at Ser139 (REF. 197), which recruits MDC1 (mediator of DNA damage checkpoint protein 1) to sites of breaks. The phosphorylation of MDC1 by ATM leads to the sequential recruitment of the E3 ubiquitin ligases RNF8 (REF. 124) and RNF168. One substrate of RNF168 is H2AK15 (histone 2A Lys15), which, together with mono- and dimethylated H4K20, serves as a platform to recruit 53BP1 (REFS 128,200), which then recruits the effector proteins RIF1 (RAP1-interacting factor 1) and PTIP (Pax transactivation domain-interacting protein), both of which bind to phosphorylated 53BP1. RIF1 functions in part by recruiting REV7 (also known as MAD2L2) to sites of breaks, where it inhibits end resection.

Figure 5. The mechanism by which DNA polymerase θ (Pol θ) promotes alternative non-homologous end joining (alt-NHEJ)

Sequence analysis of shelterin-free telomeres in Ku-deficient cells identified random nucleotide insertions at telomere fusion junctions. Subsequent genetic studies identified Pol θ as a key alt-NHEJ factor that promotes the joining of dysfunctional telomeres. Following double-strand break (DSB) formation or telomere uncapping, DNA ends are resected to create short 3′ overhangs. On the basis of in vitro experiments, genetic studies and sequence analysis of fusion junctions, Pol θ seems to be capable of extending the 3′ single-stranded DNA (ssDNA) using a combination of template-dependent as well as template-independent activities, the latter potentially mediated through a snap-back intermediate. The incorporation of random nucleotides as sites of breaks is predicted to increase the level of microhomology, thereby promoting the synapsis of opposite ends of a DSB. Annealed intermediates are then subject to fill-in synthesis by Pol θ, a step that would stabilize the duplexed DNA. Ultimately, the DNA is joined by DNA ligase 3.

Figure 6. Two independent pathways trigger telomere dysfunction in cancer

a | Telomere attrition induces telomere dysfunction and promotes gross chromosomal rearrangement due to breakage–fusion–bridge cycles (a non-reciprocal translocation is shown). Telomerase reactivation is a key event that stabilizes chromosome ends and supports the proliferation of tumours. b | Recurrent mutations in the TERT promoter are common in many cancers and seem to create a de novo binding site for the transcription factor GABP (GA-binding protein transcription factor). c | Deficiency in the shelterin subunit POT1 (protection of telomere 1) represent a novel mechanism that triggers telomere dysfunction in cancer. POT1 mutations induce telomere fragility and are associated with considerable telomere elongation. POT1 mutations also manifest in a mild chromosome fusion phenotype, which is predicted to induce chromosomal instability and augment tumour progression. d | POT1 mutations cluster primarily in its oligonucleotide-binding (OB) fold domains and are widespread across many tumour types.