DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Rif1 provides a new DNA-binding interface for the Bloom syndrome complex to maintain normal replication.

BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C-terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM-deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA-binding domain that resembles the αCTD domain of bacterial RNA polymerase α; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA-binding interface for the BLM complex to restart stalled replication forks.

A small molecule inhibitor of Pot1 binding to telomeric DNA.

Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of the regulation of these ends is a hallmark of cancer, telomeres and telomere maintenance have been prime drug targets. The universally conserved ssDNA overhang is sequence-specifically bound and regulated by Pot1 (protection of telomeres 1), and perturbation of Pot1 function has deleterious effects for proliferating cells. The specificity of the Pot1/ssDNA interaction and the key involvement of this protein in telomere maintenance have suggested directed inhibition of Pot1/ssDNA binding as an efficient means of disrupting telomere function. To explore this idea, we developed a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen for inhibitors of Pot1/ssDNA interaction. We conducted this screen with the DNA-binding subdomain of Schizosaccharomyces pombe Pot1 (Pot1pN), which confers the vast majority of Pot1 sequence-specificity and is highly similar to the first domain of human Pot1 (hPOT1). Screening a library of ∼20 000 compounds yielded a single inhibitor, which we found interacted tightly with sub-micromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red (CR), was able to competitively inhibit hPOT1 binding to telomeric DNA. Isothermal titration calorimetry and NMR chemical shift analysis suggest that CR interacts specifically with the ssDNA-binding cleft of Pot1, and that alteration of this surface disrupts CR binding. The identification of a specific inhibitor of ssDNA interaction establishes a new pathway for targeted telomere disruption.

SHARPIN Promotes Melanoma Progression via Rap1 Signaling Pathway.

SHARPIN, as a tumor-associated gene, is involved in the metastatic process of many kinds of tumors. Herein, we studied the function of Shank-associated RH domain interacting protein (SHARPIN) in melanoma metastasis and the relevant molecular mechanisms. We found that SHARPIN expression was increased in melanoma tissues and activated the process of proliferation, migration, and invasion in vitro and in vivo, resulting in a poor prognosis of the disease. Functional analysis demonstrated that SHARPIN promoted melanoma migration and invasion by regulating Ras-associated protein-1(Rap1) and its downstream pathways, including p38 and JNK/c-Jun. Rap1 activator (8-pCPT-2'-O-Me-cAMP) and inhibitor (ESI-09 and farnesylthiosalicylic acid-amide) treatments could partially rescue invasion and migration of tumor cells. Additionally, SHARPIN expression in cell lines and public datasets also indicated that molecules other than BRAF and N-RAS may contribute to SHARPIN activation. In conclusion, our broad-in-depth work suggests that SHARPIN promotes melanoma development via p38 and JNK/c-Jun pathways through upregulation of Rap1 expression.

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1.

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC(50) values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.

Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference.

Telomerase-negative cancer cells can maintain their telomeres by a recombination-mediated alternative lengthening of telomeres (ALT) process. We reported previously that sequestration of MRE11/RAD50/NBS1 complexes represses ALT-mediated telomere length maintenance, and suppresses formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are PML bodies containing telomeric DNA and telomere-binding proteins, and are observed only in a small fraction of cells within asynchronously dividing ALT-positive cell populations. Here, we report that methionine restriction caused a reversible arrest in G0/G1 phase of the cell cycle and reversible induction of APB formation in most cells within an ALT-positive population. We combined methionine restriction with RNA interference to test whether the following proteins are required for APB formation: PML body-associated proteins, PML and Sp100; telomere-associated proteins, TRF1, TRF2, TIN2 and RAP1; and DNA repair proteins, MRE11, RAD50, NBS1 and 53BP1. APB formation was not decreased by depletion of Sp100 (as reported previously) or of 53BP1, although 53BP1 partially colocalizes with APBs. Depletion of the other proteins suppressed APB formation. Because of the close linkage between ALT-mediated telomere maintenance and ability to form APBs, the eight proteins identified by this screen as being required for APB formation are also likely to be required for the ALT mechanism.

A novel small molecule that alters shelterin integrity and triggers a DNA-damage response at telomeres.

We describe a novel synthetic small molecule which shows an unprecedented stabilization of the human telomeric G-quadruplex with high selectivity relative to double-stranded DNA. We report that this compound can be used in vitro to inhibit telomerase activity and to uncap human POT1 (protection of telomeres 1) from the telomeric G-overhang. We also show that the small molecule G-quadruplex binder induces a partial alteration of shelterin through POT1 uncapping from telomeres in human HT1080 cancer cells and the presence of gammaH2AX foci colocalized at telomeres.

The G-quadruplex ligand telomestatin inhibits POT1 binding to telomeric sequences in vitro and induces GFP-POT1 dissociation from telomeres in human cells.

Telomestatin is a potent G-quadruplex ligand that specifically interacts with the 3' telomeric overhang, leading to its degradation and that induces a delayed senescence and apoptosis of cancer cells. Protection of Telomere 1 (POT1) was recently identified as a specific single-stranded telomere-binding protein involved in telomere capping and T-loop maintenance. We showed here that a telomestatin treatment inhibits POT1 binding to the telomeric overhang in vitro. The treatment of human EcR293 cells by telomestatin induces a dramatic and rapid delocalization of POT1 from its normal telomere sites but does not affect the telomere localization of the double-stranded telomere-binding protein TRF2. Thus, we propose that G-quadruplex stabilization at telomeric G-overhang inactivates POT1 telomeric function, generating a telomere dysfunction in which chromosome ends are no longer properly protected.

BRCA2 antagonizes classical and alternative nonhomologous end-joining to prevent gross genomic instability.

BRCA2-deficient cells exhibit gross genomic instability, but the underlying mechanisms are not fully understood. Here we report that inactivation of BRCA2 but not RAD51 destabilizes RPA-coated single-stranded DNA (ssDNA) structures at resected DNA double-strand breaks (DSBs) and greatly enhances the frequency of nuclear fragmentation following cell exposure to DNA damage. Importantly, these BRCA2-associated deficits are fueled by the aberrant activation of classical (c)- and alternative (alt)- nonhomologous end-joining (NHEJ), and rely on the well-defined DNA damage signaling pathway involving the pro-c-NHEJ factor 53BP1 and its downstream effector RIF1. We further show that the 53BP1-RIF1 axis promotes toxic end-joining events via the retention of Artemis at DNA damage sites. Accordingly, loss of 53BP1, RIF1, or Artemis prolongs the stability of RPA-coated DSB intermediates in BRCA2-deficient cells and restores nuclear integrity. We propose that BRCA2 antagonizes 53BP1, RIF1, and Artemis-dependent c-NHEJ and alt-NHEJ to prevent gross genomic instability in a RAD51-independent manner.

RAP1-GTPase signaling and platelet function.

Platelets are critical for hemostasis, i.e., the body's ability to prevent blood loss at sites of vascular injury. They patrol the vasculature in a quiescent, non-adhesive state for approximately 10 days, after which they are removed from circulation by phagocytic cells of the reticulo-endothelial system. At sites of vascular injury, they promptly shift to an activated, adhesive state required for the formation of a hemostatic plug. The small GTPase RAP1 is a critical regulator of platelet adhesiveness. Our recent studies demonstrate that the antagonistic balance between the RAP1 regulators, CalDAG-GEFI and RASA3, is critical for the modulation of platelet adhesiveness, both in circulation and at sites of vascular injury. The RAP1 activator CalDAG-GEFI responds to small changes in the cytoplasmic calcium concentration and thus provides sensitivity and speed to the activation response, essential for efficient platelet adhesion under conditions of hemodynamic shear stress. The RAP1 inhibitor RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI-dependent RAP1 activation. Upon cellular stimulation, it is turned off by P2Y12 signaling to enable sustained RAP1 activation, required for the formation of a stable hemostatic plug. This review will summarize important studies that elucidated the signaling pathways that control RAP1 activation in platelets.

Rap1 induces cytokine production in pro-inflammatory macrophages through NFκB signaling and is highly expressed in human atherosclerotic lesions.

Repressor activator protein 1 (Rap1) is essential for maintaining telomere length and structural integrity, but it also exerts other non-telomeric functions. The present study tested the hypothesis that Rap1 is released into the cytoplasm and induces production of pro-inflammatory cytokines via nuclear factor kappa B (NFκB) signaling in macrophages, a cell type involved in the development and progression of atherosclerotic lesions. Western blotting analysis confirmed that Rap1 was present in the cytoplasm of differentiated human monocytic leukemia cells (THP-1, a macrophage-like cell line). Co-immunoprecipitation assay revealed a direct interaction between Rap1 and I kappa B kinase (IKK). Knockdown of Rap1 suppressed lipopolysaccharide-mediated activation of NFκB, and phosphorylation of inhibitor of kappa B α (IκBα) and p65 in THP-1 macrophages. The reduction of NFκB activity was paralleled by a decreased production of NFκB-dependent pro-inflammatory cytokines and an increased expression of IκBα (native NFκB inhibitor) in various macrophage models with pro-inflammatory phenotype, including THP-1, mouse peritoneal macrophages and bone marrow-derived M1 macrophages. These changes were observed selectively in pro-inflammatory macrophages but not in bone marrow-derived M2 macrophages (with an anti-inflammatory phenotype), mouse lung endothelial cells, human umbilical vein endothelial cells or human aortic smooth muscle cells. Immunostaining revealed that Rap1 was localized mainly in macrophage-rich areas in human atherosclerotic plaques and that the presence of Rap1 was positively correlated with the advancement of the disease process. In pro-inflammatory macrophages, Rap1 promotes cytokine production via NFκB activation favoring a pro-inflammatory environment which may contribute to the development and progression of atherosclerosis.

The leukemia-associated Rho guanine nucleotide exchange factor LARG is required for efficient replication stress signaling.

We previously identified and characterized TELO2 as a human protein that facilitates efficient DNA damage response (DDR) signaling. A subsequent yeast 2-hybrid screen identified LARG; Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (also known as Arhgef12), as a potential novel TELO2 interactor. LARG was previously shown to interact with Pericentrin (PCNT), which, like TELO2, is required for efficient replication stress signaling. Here we confirm interactions between LARG, TELO2 and PCNT and show that a sub-set of LARG co-localizes with PCNT at the centrosome. LARG-deficient cells exhibit replication stress signaling defects as evidenced by; supernumerary centrosomes, reduced replication stress-induced γH2AX and RPA nuclear foci formation, and reduced activation of the replication stress signaling effector kinase Chk1 in response to hydroxyurea. As such, LARG-deficient cells are sensitive to replication stress-inducing agents such as hydroxyurea and mitomycin C. Conversely we also show that depletion of TELO2 and the replication stress signaling kinase ATR leads to RhoA signaling defects. These data therefore reveal a level of crosstalk between the RhoA and DDR signaling pathways. Given that mutations in both ATR and PCNT can give rise to the related primordial dwarfism disorders of Seckel Syndrome and Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) respectively, which both exhibit defects in ATR-dependent checkpoint signaling, these data also raise the possibility that mutations in LARG or disruption to RhoA signaling may be contributory factors to the etiology of a sub-set of primordial dwarfism disorders.

Novel anticancer strategy aimed at targeting shelterin complexes by the induction of structural changes in telomeric DNA: hitting two birds with one stone.

The ends of chromosomes in mammals are composed of telomeric DNA containing TTAGGG repeats, which bind specific proteins called shelterins. This telomeric DNA together with shelterins form a cap that protects the ends of chromosomes from being recognized as sites of DNA damage and from chromosomal fusions. Many very successful antitumor drugs used in the treatment of cancer patients bind to DNA, some of them with a prominent sequence specificity leads to changes in DNA structure and integrity. We propose a new target for antitumor drugs where small molecule ligands can bind to telomeric DNA and induce specific structural changes. These changes would lead to a selective interference with the formation of telomeric DNA-shelterin complexes, especially involving TRF1 and TRF2 proteins, as these proteins bind double-stranded telomeric DNA in a sequence- and structure-dependent manner. The rationale of the proposed therapeutic strategy is further justified by the fact that tumor cells have relatively short telomeres and frequently de-regulated shelterin expression and/or functionality. Thus uncapping of chromosome ends by DNA binding compounds which disrupt DNA-shelterin complexes can ultimately induce selective cytotoxic effect in tumor cells. Possible implications for rational design of new antitumor drugs which interfere with telomeric DNA structure and formation of DNA-shelterin complexes are discussed.

Rap1 potentiates endothelial cell junctions by spatially controlling myosin II activity and actin organization.

Reorganization of the actin cytoskeleton is responsible for dynamic regulation of endothelial cell (EC) barrier function. Circumferential actin bundles (CAB) promote formation of linear adherens junctions (AJs) and tightening of EC junctions, whereas formation of radial stress fibers (RSF) connected to punctate AJs occurs during junction remodeling. The small GTPase Rap1 induces CAB formation to potentiate EC junctions; however, the mechanism underlying Rap1-induced CAB formation remains unknown. Here, we show that myotonic dystrophy kinase-related CDC42-binding kinase (MRCK)-mediated activation of non-muscle myosin II (NM-II) at cell-cell contacts is essential for Rap1-induced CAB formation. Our data suggest that Rap1 induces FGD5-dependent Cdc42 activation at cell-cell junctions to locally activate the NM-II through MRCK, thereby inducing CAB formation. We further reveal that Rap1 suppresses the NM-II activity stimulated by the Rho-ROCK pathway, leading to dissolution of RSF. These findings imply that Rap1 potentiates EC junctions by spatially controlling NM-II activity through activation of the Cdc42-MRCK pathway and suppression of the Rho-ROCK pathway.

A mammalian KASH domain protein coupling meiotic chromosomes to the cytoskeleton.

Chromosome pairing is an essential meiotic event that ensures faithful haploidization and recombination of the genome. Pairing of homologous chromosomes is facilitated by telomere-led chromosome movements and formation of a meiotic bouquet, where telomeres cluster to one pole of the nucleus. In metazoans, telomere clustering is dynein and microtubule dependent and requires Sun1, an inner nuclear membrane protein. Here we provide a functional analysis of KASH5, a mammalian dynein-binding protein of the outer nuclear membrane that forms a meiotic complex with Sun1. This protein is related to zebrafish futile cycle (Fue), a nuclear envelope (NE) constituent required for pronuclear migration. Mice deficient in this Fue homologue are infertile. Males display meiotic arrest in which pairing of homologous chromosomes fails. These findings demonstrate that telomere attachment to the NE is insufficient to promote pairing and that telomere attachment sites must be coupled to cytoplasmic dynein and the microtubule system to ensure meiotic progression.

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in.

Telomere maintenance is critical for genome stability. The newly-identified Ctc1/Stn1/Ten1 complex is important for telomere maintenance, though its precise role is unclear. We report here that depletion of hStn1 induces catastrophic telomere shortening, DNA damage response, and early senescence in human somatic cells. These phenotypes are likely due to the essential role of hStn1 in promoting efficient replication of lagging-strand telomeric DNA. Downregulation of hStn1 accumulates single-stranded G-rich DNA specifically at lagging-strand telomeres, increases telomere fragility, hinders telomere DNA synthesis, as well as delays and compromises telomeric C-strand synthesis. We further show that hStn1 deficiency leads to persistent and elevated association of DNA polymerase α (polα) to telomeres, suggesting that hStn1 may modulate the DNA synthesis activity of polα rather than controlling the loading of polα to telomeres. Additionally, our data suggest that hStn1 is unlikely to be part of the telomere capping complex. We propose that the hStn1 assists DNA polymerases to efficiently duplicate lagging-strand telomeres in order to achieve complete synthesis of telomeric DNA, therefore preventing rapid telomere loss.

Human CST has independent functions during telomere duplex replication and C-strand fill-in.

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here, we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a replication factor that solves diverse replication-associated problems.

EGFR-dependent pancreatic carcinoma cell metastasis through Rap1 activation.

Tyrosine kinase receptors have an essential role in various aspects of tumor progression. In particular, epidermal growth factor receptor (EGFR) and its ligands have been implicated in the growth and dissemination of a wide array of human carcinomas. Here, we describe an EGFR-mediated signaling pathway that regulates human pancreatic carcinoma cell invasion and metastasis, yet does not influence the growth of primary tumors. In fact, ligation/activation of EGFR induces Src-dependent phosphorylation of two critical tyrosine residues of p130CAS, leading to the assembly of a Crk-associated substrate (CAS)/Nck1 complex that promotes Ras-associated protein-1 (Rap1) signaling. Importantly, GTP loading of Rap1 is specifically required for pancreatic carcinoma cell migration on vitronectin but not on collagen. Furthermore, Rap1 activation is required for EGFR-mediated metastasis in vivo without impacting primary tumor growth. These findings identify a molecular pathway that promotes the invasive/metastatic properties of human pancreatic carcinomas driven by EGFR.

Rap1GAP alters leukemia cell differentiation, apoptosis and invasion in vitro.

Rap1GAP which regulates the GTP-GDP form switch of Rap1 is a member of the GTPase-activating protein (GAP) family and has recently received substantial attention. Rap1GAP is thought of as a putative tumor suppressor gene and plays an important role in human tumor progression including pancreatic cancer, thyroid cancer and melanoma. In the current study, we found that the expression of Rap1GAP was lower in acute myeloid leukemia (AML) patients compared to non-malignant blood disease patients. The expression of Rap1GAP was also low in HL-60, NB4, U937 and SHI-1 myeloid leukemia cell lines. Upregulated Rap1GAP in NB4 and HL-60 cells promoted cell differentiation induced by ATRA or TPA compared to the empty vector control cells. Furthermore, Rap1GAP-transfected cells also showed a higher rate of apoptosis in response to arsenic trioxide compared to the control counterpart cells. In addition, we found that increased expression of Rap1GAP promoted leukemia cell invasion may be due to matrix metalloproteinase 9 (MMP9). In conclusion, these results demonstrated that Rap1GAP promoted leukemia cell differentiation and apoptosis, but increased leukemia cell invasion in vitro.

Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast.

Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Delta survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Delta trt1Delta cells are able to stably maintain telomeres. Thus, taz1Delta trt1Delta cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Delta trt1Delta cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Delta trt1Delta cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.