RESUMEN
Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.
Asunto(s)
Antígenos Nucleares/metabolismo , ADN Ligasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígenos Nucleares/genética , Línea Celular , Supervivencia Celular , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Autoantígeno Ku , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Rayos UltravioletaRESUMEN
The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.
Asunto(s)
Proteína Quinasa Activada por ADN/inmunología , Proteínas de Unión al ADN/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Inmunidad Innata , Proteínas Nucleares/inmunología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Proteínas Virales/inmunología , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/inmunología , Antígenos Nucleares/metabolismo , Línea Celular , Proteína Quinasa Activada por ADN/biosíntesis , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Autoantígeno Ku , Ratones Endogámicos BALB C , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Unión Proteica , Vaccinia/genética , Vaccinia/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The synapsis of DNA ends is a critical step for the repair of double-strand breaks by non-homologous end joining (NHEJ). This is performed by a multicomponent protein complex assembled around Ku70-Ku80 heterodimers and regulated by accessory factors, including long non-coding RNAs, through poorly understood mechanisms. Here, we use magnetic tweezers to investigate the contributions of core NHEJ proteins and APLF and lncRNA NIHCOLE to DNA synapsis. APLF stabilizes DNA end bridging and, together with Ku70-Ku80, establishes a minimal complex that supports DNA synapsis for several minutes under piconewton forces. We find the C-terminal acidic region of APLF to be critical for bridging. NIHCOLE increases the dwell time of the synapses by Ku70-Ku80 and APLF. This effect is further enhanced by a small and structured RNA domain within NIHCOLE. We propose a model where Ku70-Ku80 can simultaneously bind DNA, APLF, and structured RNAs to promote the stable joining of DNA ends.
Asunto(s)
ARN Largo no Codificante , ARN Largo no Codificante/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Roturas del ADN de Doble Cadena , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Reparación del ADN por Unión de Extremidades , ADN/metabolismo , Reparación del ADNRESUMEN
Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 - Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA.
Asunto(s)
Proteínas de Unión al ADN , Virus Vaccinia , Virus Vaccinia/genética , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku/metabolismo , ADN/metabolismo , Proteína Quinasa Activada por ADN/metabolismoRESUMEN
BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response; however, selective small molecule inhibitors of defined mechanism are currently lacking. Here, we identify and characterise a specific inhibitor of BLM's ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.
Asunto(s)
RecQ Helicasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , ADN/metabolismo , ADN Cruciforme , ADN de Cadena Simple , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli , Ensayos Analíticos de Alto Rendimiento , Humanos , RecQ Helicasas/metabolismoRESUMEN
Long noncoding RNAs (lncRNA) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the nonhomologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of blunt-ended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability. SIGNIFICANCE: This study characterizes the role of lncRNA NIHCOLE in DNA repair and cellular fitness in HCC, thus implicating it as a therapeutic target.See related commentary by Barcena-Varela and Lujambio, p. 4899.
Asunto(s)
Carcinoma Hepatocelular/genética , Roturas del ADN de Doble Cadena , Neoplasias Hepáticas/genética , ARN Largo no Codificante/genética , Biomarcadores de Tumor , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidad , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidad , Modelos Biológicos , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Pronóstico , ARN Largo no Codificante/químicaRESUMEN
The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg(2+) bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerization of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving alpha1, loop beta5alpha5 and N terminus of alpha5 elements, also seen in the complex of PhoB RD with Mg(2+), in which helix alpha4 highly increases its flexibility. PhoB RD in complex with Mg(2+) and BeF(3) (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix alpha4 and shows another interaction involving alpha4, beta5 and alpha5 segments. We have selected a series of constitutively active PhoB mutants (PhoB(CA)) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoB(CA) RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix alpha4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Mutación , Estructura Terciaria de Proteína , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Escherichia coli/genética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Variolin B is a rare marine alkaloid that showed promising anti-cancer activity soon after its isolation. It acts as a cyclin-dependent kinase inhibitor, although the precise mechanism through which it exerts the cytotoxic effects is still unknown. The crystal structure of a variolin B bound to a DNA forming a pseudo-Holliday junction shows that this compound can also contribute, through intercalative binding, to either the formation or stabilization of multi-stranded DNA forms.
Asunto(s)
Antineoplásicos/química , Compuestos Aza/química , ADN/química , Sustancias Intercalantes/química , Pirimidinas/química , Cristalografía por Rayos X , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ.
Asunto(s)
Antígenos Nucleares/metabolismo , Secuencia Conservada , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Roturas del ADN de Doble Cadena , Daño del ADN , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Humanos , Autoantígeno Ku , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , RecQ Helicasas/química , RecQ Helicasas/metabolismo , Helicasa del Síndrome de WernerRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP1) is a primary DNA damage sensor whose (ADP-ribose) polymerase activity is acutely regulated by interaction with DNA breaks. Upon activation at sites of DNA damage, PARP1 modifies itself and other proteins by covalent addition of long, branched polymers of ADP-ribose, which in turn recruit downstream DNA repair and chromatin remodeling factors. PARP1 recognizes DNA damage through its N-terminal DNA-binding domain (DBD), which consists of a tandem repeat of an unusual zinc-finger (ZnF) domain. We have determined the crystal structure of the human PARP1-DBD bound to a DNA break. Along with functional analysis of PARP1 recruitment to sites of DNA damage in vivo, the structure reveals a dimeric assembly whereby ZnF1 and ZnF2 domains from separate PARP1 molecules form a strand-break recognition module that helps activate PARP1 by facilitating its dimerization and consequent trans-automodification.