RESUMEN
The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.
Asunto(s)
Síndromes de Tricotiodistrofia , Animales , Ratones , Intrones/genética , Síndromes de Tricotiodistrofia/genética , ARN Nucleotidiltransferasas/genética , Empalme del ARNRESUMEN
Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here, we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration-dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the end of RAD51 filaments.
Asunto(s)
Proteínas de Unión al ADN , Recombinasa Rad51 , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/metabolismo , Microscopía por Crioelectrón , Nucleoproteínas/metabolismo , ADN de Cadena Simple , Replicación del ADNRESUMEN
Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.
Asunto(s)
Hidrolasas Diéster Fosfóricas , Humanos , Biología Computacional/métodos , Cristalografía por Rayos X , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Motivos de Unión al ARN/genéticaRESUMEN
Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.
Asunto(s)
Adenosina Trifosfatasas , ADN Polimerasa theta , Neoplasias , Novobiocina , Humanos , Adenosina Trifosfatasas/metabolismo , Replicación del ADN , ADN de Cadena Simple , ADN Polimerasa Dirigida por ADN/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Novobiocina/farmacologíaRESUMEN
The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.
Asunto(s)
Reparación del ADN , Proteína de Replicación A , Proteína de la Xerodermia Pigmentosa del Grupo A , ADN/metabolismo , Daño del ADN , Unión Proteica , Dominios Proteicos , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.
Asunto(s)
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Síndrome de Cockayne/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Inestabilidad Genómica , Recombinación Homóloga , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Síndrome de Cockayne/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Genoma Humano , Células HeLa , Humanos , Ratones , Proteínas Nucleares/metabolismo , Fosforilación , Recombinasa Rad51/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.
Asunto(s)
ADN Ligasa (ATP)/metabolismo , Reparación del ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Cromatografía en Gel , Cristalografía por Rayos X , ADN Ligasa (ATP)/química , Dimerización , Humanos , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos , Mutación , Mutación Missense , Coloración Negativa , Mutación Puntual , Conformación Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/química , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genéticaRESUMEN
Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.
Asunto(s)
Síndrome de Cockayne/genética , Proteínas de Unión al ADN/química , Endonucleasas/química , Proteínas Nucleares/química , Mutación Puntual , Factores de Transcripción/química , Xerodermia Pigmentosa/genética , Sitios de Unión , Secuencia Conservada , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Estabilidad de Enzimas , Humanos , Simulación de Dinámica Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII170-755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.
Asunto(s)
ADN Ligasa (ATP) , Proteínas de Unión a Poli-ADP-Ribosa , Dominio Catalítico , Daño del ADN , Reparación del ADN , Humanos , FosforilaciónRESUMEN
Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Autoantígeno Ku/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Unión Proteica , Multimerización de ProteínaRESUMEN
Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.
Asunto(s)
Factor 1 de Ensamblaje de la Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Oxidación-Reducción , Estrés Oxidativo , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Daño del ADN/efectos de la radiación , ADN Glicosilasas/metabolismo , Glucosa Oxidasa/metabolismo , Histonas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos , Unión Proteica , Radiación Ionizante , Especies Reactivas de Oxígeno , Factores de TranscripciónRESUMEN
Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3Î-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Humanos , Fosforilación , Rayos X , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.
Asunto(s)
Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP)/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku/metabolismo , Proteínas Nucleares/metabolismo , Western Blotting , Reactivos de Enlaces Cruzados , Roturas del ADN de Doble Cadena , ADN Ligasa (ATP)/química , ADN Ligasa (ATP)/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteína Quinasa Activada por ADN/química , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Inmunoprecipitación , Autoantígeno Ku/química , Autoantígeno Ku/genética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Conformación Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.
Asunto(s)
ADN Viral/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Ubiquitinación , Sustitución de Aminoácidos , Animales , ADN Circular/química , Proteína del Grupo de Complementación L de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Proteína 2 Inhibidora de la Diferenciación/química , Nucleosomas/química , Plásmidos/química , Unión Proteica , Células Sf9 , Spodoptera , Especificidad por SustratoRESUMEN
Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.
Asunto(s)
Proteínas de Ciclo Celular/química , Reparación del ADN , ADN/química , Exonucleasas/química , Endonucleasas de ADN Solapado/química , Antígeno Nuclear de Célula en Proliferación/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/genética , ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Exonucleasas/genética , Exonucleasas/metabolismo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Humanos , Microscopía Electrónica , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Conformación de Ácido Nucleico , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Meiosis , Proteínas Nucleares/metabolismo , Recombinasas/metabolismo , Animales , Cromatina/metabolismo , Emparejamiento Cromosómico , Proteínas de Unión al ADN/aislamiento & purificación , Humanos , Masculino , Ratones , Proteínas Mutantes/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Proteínas de Unión a Fosfato , Unión Proteica , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Proteínas de Unión al ARN , Recombinasa Rad51/metabolismo , Espermatocitos/citología , Espermatocitos/metabolismoRESUMEN
Certain environmental toxins are nucleic acid damaging agents, as are many chemotherapeutics used for cancer therapy. These agents induce various adducts in DNA as well as RNA. Indeed, most of the nucleic acid adducts (>90%) formed due to these chemicals, such as alkylating agents, occur in RNA 1 . However, compared to the well-studied mechanisms for DNA alkylation repair, the biological consequences of RNA damage are largely unexplored. Here, we demonstrate that RNA damage can directly result in loss of genome integrity. Specifically, we show that a human YTH domain-containing protein, YTHDC1, regulates alkylation damage responses in association with the THO complex (THOC) 2 . In addition to its established binding to N 6-methyladenosine (m6A)-containing RNAs, YTHDC1 binds to N 1-methyladenosine (m1A)-containing RNAs upon alkylation. In the absence of YTHDC1, alkylation damage results in increased alkylation damage sensitivity and DNA breaks. Such phenotypes are fully attributable to RNA damage, since an RNA-specific dealkylase can rescue these phenotypes. These R NA d amage-induced DNA b reaks (RDIBs) depend on R-loop formation, which in turn are processed by factors involved in transcription-coupled nucleotide excision repair. Strikingly, in the absence of YTHDC1 or THOC, an RNA m1A methyltransferase targeted to the nucleus is sufficient to induce DNA breaks. Our results uncover a unique role for YTHDC1-THOC in base damage responses by preventing RDIBs, providing definitive evidence for how damaged RNAs can impact genomic integrity.
RESUMEN
XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.
Asunto(s)
Enzimas Reparadoras del ADN/química , Reparación del ADN , Proteínas de Unión al ADN/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Supervivencia Celular , Daño del ADN , ADN Ligasas/metabolismo , Regulación de la Expresión Génica , Humanos , Cinética , Microscopía Confocal/métodos , Mutación , Proteínas Nucleares/química , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Estructura Terciaria de Proteína , Treonina/química , Técnicas del Sistema de Dos Híbridos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.