N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Aptamer photoregulation in vivo

The in vivo application of aptamers as therapeutics could be improved by enhancing target-specific accumulation while minimizing off-target uptake. We designed a light-triggered system that permits spatiotemporal regulation of aptamer activity in vitro and in vivo. Cell binding by the aptamer was prevented by hybridizing the aptamer to a photo-labile complementary oligonucleotide. Upon irradiation at the tumor site, the aptamer was liberated, leading to prolonged intratumoral retention. The relative distribution of the aptamer to the liver and kidney was also significantly decreased, compared to that of the free aptamer.Aptamers are single-stranded nucleic acids that have emerged as a promising class of therapeutics owing to their relative ease of synthesis and high affinity and selectivity toward a range of targets including small molecules, proteins, viral particles, and living cells (1–6). Aptamers can fold into well-defined conformations and are more resistant to enzymatic degradation than other oligonucleotides (7–9). Aptamers have been suggested for imaging applications because their relatively small size and molecular mass (∼10 kDa) allow fast tissue penetration and clearance from blood (10, 11). The same characteristics make aptamers promising for effective delivery of diagnostic and therapeutic agents to tissues or organs. However, nonspecific accumulation of aptamers in normal tissues is undesirable (12–15) because it diminishes the proportion of aptamer that targets the desired tissue. This can adversely affect the therapeutic index of the aptamer; this may be particularly true if the aptamer is conjugated to a drug or drug delivery device. Moreover, aptamers themselves can have nonspecific toxic effects (16, 17). Ideally aptamers would achieve a high concentration in a pathological tissue of interest while maintaining low levels elsewhere. The activity of aptamers can be modulated in vivo by binding to polymers or complementary oligonucleotide sequences (18–20), but spatiotemporal regulation of aptamer activity in vivo has not been achieved, whereby activity would be enhanced in target tissues and not others. Here, we report a strategy to provide light-triggered control of aptamer function and distribution in vivo.Light is an excellent means of providing external spatiotemporal control of biological systems (21–26). Many strategies have been developed to incorporate photosensitive groups in nucleotides that can control cellular function or affect biological pathways or gene expression by light (24–29). Of particular interest, such approaches can be used to provide spatiotemporal control of gene activation (24). Here we hypothesized that light triggering can be used to achieve spatiotemporal control of binding of an aptamer injected systemically to its target tissue in vivo, which would have implications for control of delivery of therapeutic aptamers and/or conjugated drugs or drug delivery systems. We designed a photo-triggerable system whereby the aptamer of interest is inactivated by hybridization to a photo-labile complementary oligonucleotide. Upon irradiation, the complementary sequence breaks down, releasing the functional aptamer (Fig. 1). The aptamer of interest is the single-stranded DNA 26-mer aptamer AS1411 (A₁₄₁₁; sequence: 5′-GGT GGT GGT GGT TGT GGT GGT GGT GG-3′) that binds with high affinity and selectivity to nucleolin (30–32), which is overexpressed on the cell membrane of several types of cancer cells, including the 4T1 breast cancer cells used here (33–35). A₁₄₁₁ has been used for cancer targeting in vitro and in vivo (35, 36). A complementary photo-triggerable inhibitory oligonucleotide (OliP) was designed [sequence: 5′-CCA CCA//CCA CCA//CAA CCA C-3′, where // indicates photo-labile 1-(2-nitrophenyl)ethyl bonds (37); Scheme S1].Open in a separate windowFig. 1.The AS1411 aptamer (A₁₄₁₁, red DNA strand) is hybridized to a complementary oligonucleotide (OliP, green DNA strand) containing photo-cleavable bonds (black dots). The hybridized complex cannot bind cells. The A₁₄₁₁ can be released by light-triggered breakage of the OliP, allowing binding to cell surfaces.     

Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.Aptamers are artificially selected oligonucleotides that bind to molecular targets, typically proteins, with high specificity and avidity (1–3). DNA aptamers have been selected against dozens of targets for biomedical applications both as therapeutics (4, 5) and diagnostics (6, 7). Despite their widespread application, few DNA aptamer-target complex structures have been solved (8)–the best studied of which is the G-quadruplex aptamer that binds to thrombin (9–12). A DNA aptamer that binds to von Willebrand factor showed a three-stem structure of mainly B-form DNA with some noncanonical base pairing (13). Most recently, the structure of an innovative Slow Off-rate Modified Aptamer (SOMAmer) bound to platelet-derived growth factor B was solved, revealing binding via a hydrophobic surface that mimics how the factor binds to its receptor (14). Generally, the lack of DNA aptamer-target structures has limited our understanding of the mechanisms by which DNA aptamers attain their specificity (15), resulting in a bias in aptasensor development (16).Better point-of-care tests are critically needed for malaria, a disease which continues to claim more than 1 million lives globally every year (17). Antimalarial drugs have been administered presumptively to patients with fever for decades, leading to drug resistance and poor management of other febrile illness. The cost of newer, more effective treatments has led to a situation whereby improved diagnostics has become a major factor that could reduce the burden of malaria in the developing world (17). Antibody-based rapid diagnostic tests have greatly benefitted malaria management, but significant issues with cost (17) and stability in tropical climates (18) remain that are intrinsically associated with the use of protein antibodies. DNA aptamers compare favorably to antibodies for diagnostic applications (19) with particular advantages that could be critical for diagnostic tests of the developing world: thermal stability, convenient chemical synthesis, and potentially lower costs of production (16). Here, we report the crystal structure and application of a unique DNA aptamer against an established malaria pan-species diagnostic target, Plasmodium falciparum lactate dehydrogenase (PfLDH) (20), and a mechanism of molecular recognition by a distorted hairpin DNA aptamer.     

DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions

DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.Adult stem cells (SCs) are thought to be resistant to DNA damage (DD)-induced apoptosis or senescence owing to the activation of unique pro-survival and DD repair (DDR) responses (1–3). Genetic alterations that decrease DNA repair activities lead to increased DD and reduced self-renewal in SCs, suggesting that DDR is critical to preservation of SC function (1, 4, 5). DDR decreases during physiological aging, a phenomenon correlated with the accumulation of endogenous DD and decreased self-renewal in aged SCs (6–9).In differentiated cells, DD triggers a checkpoint response that leads to apoptosis or senescence and depends on activation of the tumor suppressor p53 (10). This is considered a powerful tumor-suppressor mechanism, as demonstrated by the finding that p53 is invariably inactivated in spontaneous tumors (11). After irradiation, p53 is up-regulated in populations enriched for hematopoietic, hair follicle bulge, and colon SCs (5, 12–15). Whether this is critical for activation of the DDR response and maintenance of self-renewal, why p53 induction does not result in SC apoptosis or senescence, and how tumor suppression is executed in SCs remain unclear, however. Indirect evidence indicates that the cell cycle inhibitor p21, a downstream effector of p53, might be involved in DD processing in SCs. In the absence of p21, SCs exhaust prematurely (16) and after a low radiation dose display reduced reconstitution capacity (17). Here we report our studies on the role of p53 and p21 in DD processing of highly purified hematopoietic SCs (HSCs) and mammary SCs (MaSCs).     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Role of 53BP1 oligomerization in regulating double-strand break repair

Tumor suppressor p53-binding protein 1 (53BP1) regulates the repair of dysfunctional telomeres lacking the shelterin protein TRF2 by promoting their mobility, their nonhomologous end-joining (NHEJ), and, as we show here, by blocking 5′ resection by CtIP. We report that these functions of 53BP1 required its N-terminal ATM/ATR target sites and its association with H4K20diMe, but not the BRCT domain, the GAR domain, or the binding of 53BP1 to dynein. A mutant lacking the oligomerization domain (53BP1^oligo) was only modestly impaired in promoting NHEJ of dysfunctional telomeres and showed no defect with regard to the repression of CtIP. This 53BP1^oligo allele was previously found to be unable to support class switch recombination or to promote radial chromosome formation in PARP1 inhibitor-treated Brca1-deficient cells. The data therefore support two conclusions. First, the requirements for 53BP1 in mediating NHEJ at dysfunctional telomeres and in class switch recombination are not identical. Second, 53BP1-dependent repression of CtIP at double-strand breaks (DSBs) is unlikely to be sufficient for the generation of radial chromosomes in PARP1 inhibitor-treated Brca1-deficient cells.The DNA damage response factor 53BP1 is a key regulator of the processing and repair of double-strand breaks (DSBs) (reviewed in refs. 1–3). Accumulation of 53BP1 at sites of DNA damage depends on phosphorylation of H2AX by the ATM and/or ATR kinases, binding of MDC1 to phosphorylated H2AX (γ-H2AX), and ubiquitylation of H2A and/or H2AX by MDC1-dependent ubiquitin ligases. Despite its dependence on these ATM/ATR-initiated events, 53BP1 does not bind H2AX, H2A, MDC1, or their interacting factors. Instead, 53BP1 interacts with histone H4 through an association of its tandem Tudor domain with the dimethylated form of H4 lysine 20 (H4K20Me2) (4). H4K20Me2 is a constitutive modification that has been proposed to become more accessible near sites of DNA damage due to ubiquitin-dependent removal of H4K20Me2 binding proteins, thus explaining the dependence of 53BP1 accumulation on γ-H2AX, MDC1, and ubiquitin ligases (5–7).The role of 53BP1 in DNA repair surfaced in the context of Ig class switch recombination (CSR) in which 53BP1 is essential for nonhomologous end-joining (NHEJ) of activation-induced (cytidine) deaminase-induced DSBs and has been implicated in the synapsis of DNA ends separated by as much as 200 kb (8, 9). 53BP1 also promotes NHEJ during V(D)J recombination, in particular when the recombining ends are far apart, suggesting that 53BP1 can mediate the juxtaposition of distant DNA ends and facilitate their joining (10). Similarly, 53BP1 promotes fusions of dysfunctional telomeres generated through deletion of the shelterin protein TRF2 (11). Such deprotected telomeres undergo classical-NHEJ (c-NHEJ) in G1, forming trains of fused chromosomes that can be visualized in the following metaphase (12, 13). Live-cell imaging showed that, before their joining, dysfunctional telomeres become more mobile and sample larger territories (11), as was recently also shown for other DSBs (14–16). Because this change in mobility depends on 53BP1, it was proposed that 53BP1 stimulates the fusion of telomeres by improving the chance of telomere–telomere encounters (11). How 53BP1 might promote the synapsis and/or mobility of DSBs has not been established.53BP1 also affects DNA repair through regulating DSB resection. During V(D)J recombination, the unrepaired coding ends are degraded when 53BP1 is absent (10), and 53BP1 deficiency results in frequent resection of DNA ends generated by I-SceI (17, 18). End resection is also unleashed in the absence of 53BP1 at telomeres that are deprived of all shelterin components (19). The 53BP1-controlled resection is dependent on ATM signaling and involves the CtIP nuclease (17–19).In cells lacking BRCA1 function, PARP1 inhibitors (PARPi) induce lethal radial chromosomes that are thought to result from mis-rejoined DSBs (20, 21). Deletion of 53BP1 prevents the formation of these aberrant chromosomes and rescues the lethality of Brca1 deficiency in the mouse (18, 22, 23). It has been proposed that the effect of 53BP1 in this context is due to its propensity to block resection of DSBs thus preventing formation of the 3′ extensions needed to initiate homologous recombination and favoring NHEJ.Using dysfunctional telomeres as an experimental setting, we determined which domains of 53BP1 are involved in repressing the CtIP-dependent 5′ end resection taking place in S/G2 and the induction of chromatin mobility and NHEJ in G1. This analysis revealed that oligomerization of 53BP1, although crucial for CSR, plays a lesser role in the joining of telomeres, indicating mechanistic differences between these two 53BP1-dependent NHEJ pathways. Furthermore, we find that oligomerization of 53BP1 is not required for the repression of CtIP-mediated 5′ end resection at telomeres. Because the oligomerization mutant was previously shown to be defective in radial chromosome formation in PARPi-treated Brca1 null cells (24), we infer that it is unlikely that CtIP inhibition is the sole determinant of this attribute of 53BP1.