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1.
Mol Cell ; 83(5): 698-714.e4, 2023 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-36724784

RESUMEN

Non-homologous end joining is the major double-strand break repair (DSBR) pathway in mammals. DNA-PK is the hub and organizer of multiple steps in non-homologous end joining (NHEJ). Recent high-resolution structures show how two distinct NHEJ complexes "synapse" two DNA ends. One complex includes a DNA-PK dimer mediated by XLF, whereas a distinct DNA-PK dimer forms via a domain-swap mechanism where the C terminus of Ku80 from one DNA-PK protomer interacts with another DNA-PK protomer in trans. Remarkably, the distance between the two synapsed DNA ends in both dimers is the same (∼115 Å), which matches the distance observed in the initial description of an NHEJ long-range synaptic complex. Here, a mutational strategy is used to demonstrate distinct cellular function(s) of the two dimers: one promoting fill-in end processing, while the other promotes DNA end resection. Thus, the specific DNA-PK dimer formed (which may be impacted by DNA end structure) dictates the mechanism by which ends will be made ligatable.


Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Animales , Proteínas de Unión al ADN/genética , Subunidades de Proteína/metabolismo , Reparación del ADN por Unión de Extremidades , Reparación del ADN , ADN/genética , Proteína Quinasa Activada por ADN/genética , Autoantígeno Ku/genética , Mamíferos/metabolismo
2.
Mol Cell ; 82(1): 177-189.e4, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34936881

RESUMEN

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.


Asunto(s)
Daño del ADN , Reparación del ADN por Unión de Extremidades , ADN de Neoplasias/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Neoplasias del Cuello Uterino/enzimología , ADN de Neoplasias/genética , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Activación Enzimática , Femenino , Células HEK293 , Células HeLa , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Conformación de Ácido Nucleico , Fosforilación , Unión Proteica , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología
3.
Mol Cell ; 81(4): 801-810.e3, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33385326

RESUMEN

DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKINs) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 Å at best. Here, we report the cryoelectron microscopy (cryo-EM) structures of DNA-PKcs (DNA-PK catalytic subunit) bound to a DNA end or complexed with Ku70/80 and DNA in both inactive and activated forms at resolutions of 3.7 Å overall and 3.2 Å for FATKINs. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.


Asunto(s)
Reparación del ADN por Unión de Extremidades , Proteína Quinasa Activada por ADN/química , Autoantígeno Ku/química , Complejos Multiproteicos/química , Microscopía por Crioelectrón , Proteína Quinasa Activada por ADN/genética , Activación Enzimática , Células HEK293 , Células HeLa , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura
4.
Mol Cell ; 70(2): 358-370.e4, 2018 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-29628308

RESUMEN

To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.


Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación V(D)J , Sitios de Unión , Catálisis , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/genética , ADN/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/ultraestructura , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad
5.
Nature ; 518(7540): 507-11, 2015 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-25707801

RESUMEN

V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2 Å resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Homeodominio/química , VDJ Recombinasas/química , Animales , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Modelos Moleculares , Mutación/genética , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Inmunodeficiencia Combinada Grave/genética , Transposasas/química , VDJ Recombinasas/metabolismo , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética
6.
Proc Natl Acad Sci U S A ; 112(28): 8579-83, 2015 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-26124138

RESUMEN

The variable domains of Ig and T-cell receptor genes in vertebrates are assembled from gene fragments by the V(D)J recombination process. The RAG1-RAG2 recombinase (RAG1/2) initiates this recombination by cutting DNA at the borders of recombination signal sequences (RSS) and their neighboring gene segments. The RAG1 protein is also known to contain a ubiquitin E3 ligase activity, located in an N-terminal region that is not strictly required for the basic recombination reaction but helps to regulate recombination. The isolated E3 ligase domain was earlier shown to ubiquitinate one site in a neighboring RAG1 sequence. Here we show that autoubiquitination of full-length RAG1 at this specific residue (K233) results in a large increase of DNA cleavage by RAG1/2. A mutational block of the ubiquitination site abolishes this effect and inhibits recombination of a test substrate in mouse cells. Thus, ubiquitination of RAG1, which can be promoted by RAG1's own ubiquitin ligase activity, plays a significant role in governing the level of V(D)J recombination activity.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Ubiquitinación , Recombinación V(D)J , Animales , División del ADN , Ratones
7.
Mol Cell ; 35(2): 217-27, 2009 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-19647518

RESUMEN

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación Genética/fisiología , VDJ Recombinasas/fisiología , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/ultraestructura , Proteínas de Homeodominio/química , Proteínas de Homeodominio/ultraestructura , Inmunohistoquímica , Proteínas de Unión a Maltosa , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Modelos Moleculares , Coloración Negativa , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/análisis , VDJ Recombinasas/química , VDJ Recombinasas/ultraestructura
8.
J Biol Chem ; 290(23): 14618-25, 2015 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-25903130

RESUMEN

Mammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 paired complex is active in the presence of Mg(2+), the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex. We report here the purification and initial crystallization of the RAG1/2 signal-end complex for atomic-resolution structure elucidation. Strict pairing of the 12RSS and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.


Asunto(s)
División del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Cristalización , ADN/química , ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Expresión Génica , Células HEK293 , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Ratones , Multimerización de Proteína , Recombinación V(D)J
9.
Proc Natl Acad Sci U S A ; 110(24): 9873-8, 2013 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-23716691

RESUMEN

The ability to sense metabolic stress is critical for successful cellular adaptation. In eukaryotes, the AMP-activated protein kinase (AMPK), a highly conserved serine/threonine kinase, functions as a critical metabolic sensor. AMPK is activated by the rising ADP/ATP and AMP/ATP ratios during conditions of energy depletion and also by increasing intracellular Ca(2+). In response to metabolic stress, AMPK maintains energy homeostasis by phosphorylating and regulating proteins that are involved in many physiological processes including glucose and fatty acid metabolism, transcription, cell growth, mitochondrial biogenesis, and autophagy. Evidence is mounting that AMPK also plays a role in a number of pathways unrelated to energy metabolism. Here, we identify the recombination-activating gene 1 protein (RAG1) as a substrate of AMPK. The RAG1/RAG2 complex is a lymphoid-specific endonuclease that catalyzes specific DNA cleavage during V(D)J recombination, which is required for the assembly of the Ig and T-cell receptor genes of the immune system. AMPK directly phosphorylates RAG1 at serine 528, and the phosphorylation enhances the catalytic activity of the RAG complex, resulting in increased cleavage of oligonucleotide substrates in vitro, or increased recombination of an extrachromosomal substrate in a cellular assay. Our results suggest that V(D)J recombination can be regulated by AMPK activation, providing a potential new link between metabolic stress and development of B and T lymphocytes.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas de Homeodominio/metabolismo , Serina/metabolismo , Recombinación V(D)J , Secuencia de Aminoácidos , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Immunoblotting , Ratones , Ratones Noqueados , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Fosforilación , Homología de Secuencia de Aminoácido , Serina/genética , Especificidad por Sustrato
10.
Proc Natl Acad Sci U S A ; 107(52): 22487-92, 2010 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-21149691

RESUMEN

Gene assembly of the variable domain of antigen receptors is initiated by DNA cleavage by the RAG1-RAG2 protein complex at sites flanking V, D, and J gene segments. Double-strand breaks are produced via a single-strand nick that is converted to a hairpin end on coding DNA and a blunt end on the neighboring recombination signal sequence. We demonstrate that the C-terminal regions of purified murine RAG1 (aa 1009-1040) and RAG2 (aa 388-520, including a plant homeodomain [PHD domain]) collaborate to inhibit the hairpinning stage of DNA cleavage. The C-terminal region of RAG2 stabilizes the RAG1/2 heterotetramer but destabilizes the RAG-DNA precleavage complex. This destabilization is reversed by binding of the PHD domain to a histone H3 peptide trimethylated on lysine 4 (H3K4me3). The addition of H3K4me3 likewise alleviates the RAG1/RAG2 C-terminus-mediated inhibition of hairpinning and the PHD-mediated inhibition of transposition activity. Thus a negative regulatory function of the noncore regions of RAG1/2 limits the RAG endonuclease activity in the absence of an activating methylated histone tail bound to the complex.


Asunto(s)
División del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación Genética , Exones VDJ/genética , Animales , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Epigenómica , Células HEK293 , Histonas/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Lisina/metabolismo , Metilación , Ratones , Mutación , Unión Proteica , Multimerización de Proteína , Transducción de Señal
11.
Curr Opin Struct Biol ; 71: 79-86, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34245989

RESUMEN

RAG1/2 (RAG) is an RNH-type DNA recombinase specially evolved to initiate V(D)J gene rearrangement for generating the adaptive immune response in jawed vertebrates. After decades of frustration with little mechanistic understanding of RAG, the crystal structure of mouse RAG recombinase opened the flood gates in early 2015. Structures of three different chordate RAG recombinases, including protoRAG, and the evolutionarily preceding transib transposase have been determined in complex with various DNA substrates. Biochemical studies along with the abundant structural data have shed light on how RAG has evolved from an ordinary transposase to a specialized recombinase in initiating gene rearrangement. RAG has also become one of the best characterized RNH-type recombinases, illustrating how a single active site can cleave the two antiparallel DNA strands of a double helix.


Asunto(s)
Proteínas de Homeodominio , Recombinasas , Inmunidad Adaptativa , Animales , Genes RAG-1 , Proteínas de Homeodominio/genética , Ratones , Recombinasas/genética , Recombinación V(D)J
12.
J Immunol ; 181(11): 7825-34, 2008 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19017972

RESUMEN

V(H) replacement is a form of IgH chain receptor editing that is believed to be mediated by recombinase cleavage at cryptic recombination signal sequences (cRSS) embedded in V(H) genes. Whereas there are several reports of V(H) replacement in primary and transformed human B cells and murine models, it remains unclear whether V(H) replacement contributes to the normal human B cell repertoire. We identified V(H)-->V(H)(D)J(H) compound rearrangements from fetal liver, fetal bone marrow, and naive peripheral blood, all of which involved invading and recipient V(H)4 genes that contain a cryptic heptamer, a 13-bp spacer, and nonamer in the 5' portion of framework region 3. Surprisingly, all pseudohybrid joins lacked the molecular processing associated with typical V(H)(D)J(H) recombination or nonhomologous end joining. Although inefficient compared with a canonical recombination signal sequences, the V(H)4 cRSS was a significantly better substrate for in vitro RAG-mediated cleavage than the V(H)3 cRSS. It has been suggested that activation-induced cytidine deamination (AICDA) may contribute to V(H) replacement. However, we found similar secondary rearrangements using V(H)4 genes in AICDA-deficient human B cells. The data suggest that V(H)4 replacement in preimmune human B cells is mediated by an AICDA-independent mechanism resulting from inefficient but selective RAG activity.


Asunto(s)
Citidina Desaminasa/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Modelos Biológicos , Hipermutación Somática de Inmunoglobulina/fisiología , VDJ Recombinasas/inmunología , Animales , Línea Celular Transformada , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Señales de Clasificación de Proteína/genética , Recombinación Genética/genética , Recombinación Genética/inmunología , VDJ Recombinasas/genética
13.
Nat Struct Mol Biol ; 27(2): 127-133, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32015553

RESUMEN

The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination, but RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in developing lymphocytes, we determined the structure of a DNA-strand transfer complex of mouse RAG at 3.1-Å resolution. The target DNA is a T form (T for transpositional target), which contains two >80° kinks towards the minor groove, only 3 bp apart. RAG2, a late evolutionary addition in V(D)J recombination, appears to enforce the sharp kinks and additional inter-segment twisting in target DNA and thus attenuates unwanted transposition. In contrast to strand transfer complexes of genuine transposases, where severe kinks occur at the integration sites of target DNA and thus prevent the reverse reaction, the sharp kink with RAG is 1 bp away from the integration site. As a result, RAG efficiently catalyzes the disintegration reaction that restores the RSS (donor) and target DNA.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Microscopía por Crioelectrón , ADN/química , División del ADN , Proteínas de Unión al ADN/química , Células HEK293 , Proteínas de Homeodominio/química , Humanos , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica
14.
Nat Struct Mol Biol ; 27(2): 119-126, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32015552

RESUMEN

A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating interstrand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin-DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Secuencia de Bases , Dominio Catalítico , Microscopía por Crioelectrón , ADN/química , Proteínas de Unión al ADN/química , Células HEK293 , Proteínas de Homeodominio/química , Humanos , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica
15.
Oncotarget ; 7(11): 12962-74, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26887046

RESUMEN

Mutations in the Recombination Activating Gene 1 (RAG1) can cause a wide variety of clinical and immunological phenotypes in humans, ranging from absence of T and B lymphocytes to occurrence of autoimmune manifestations associated with expansion of oligoclonal T cells and production of autoantibodies. Although the mechanisms underlying this phenotypic heterogeneity remain poorly understood, some genotype-phenotype correlations can be made. Currently, mouse models of Rag deficiency are restricted to RAG1-/- mice and to knock-in models carrying severe missense mutations. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system is a novel and powerful gene-editing strategy that permits targeted introduction of DNA double strand breaks with high efficiency through simultaneous delivery of the Cas9 endonuclease and a guide RNA (gRNA). Here, we report on CRISPR-based, single-step generation and characterization of mutant mouse models in which gene editing was attempted around residue 838 of RAG1, a region whose functional role had not been studied previously.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Proteínas de Homeodominio/genética , Ratones Mutantes/genética , Animales , Ratones , Mutagénesis Sitio-Dirigida/métodos , Cigoto
16.
J Mol Biol ; 334(2): 215-28, 2003 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-14607114

RESUMEN

The XRCC4 protein is of critical importance for the repair of broken chromosomal DNA by non-homologous end joining (NHEJ). The absence of XRCC4 abolishes chromosomal NHEJ almost completely. One reason for this severe phenotype is that XRCC4 binds and modulates the stability and activity of the NHEJ-specific ligase, DNA ligase IV. XRCC4 in solution is in equilibrium between the dimeric and tetrameric forms. Previous structural studies have shown that the interface between dimers is located in the same region as that implicated in DNA ligase IV interaction. With the use of equilibrium sedimentation analysis, we show here that only the XRCC4 dimer can associate with DNA ligase IV, forming a monodisperse complex of 2:1 stoichiometry in solution. In addition, physical analysis of XRCC4/DNA ligase IV complex formation, combined with mutational analysis of XRCC4, indicates that tetramerization and DNA ligase IV binding are mutually exclusive. We propose that the putative function of the XRCC4 tetramer is distinct from its DNA ligase IV-associated function.


Asunto(s)
ADN Ligasas/metabolismo , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Animales , Células CHO , Cricetinae , ADN Ligasa (ATP) , Proteínas de Unión al ADN/genética , Reordenamiento Génico/genética , Humanos , Mutación , Radiación Ionizante
18.
Nat Struct Mol Biol ; 19(1): 72-8, 2011 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-22179786

RESUMEN

DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutSα is well characterized. We report here crystal structures of human MutSß in complex with DNA containing insertion-deletion loops (IDL) of two, three, four or six unpaired nucleotides. In contrast to eukaryotic MutSα and bacterial MutS, which bind the base of a mismatched nucleotide, MutSß binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flipped out into the major groove and partially exposed to solvent. A normal downstream base pair can become unpaired; a single unpaired base can thereby be converted to an IDL of two nucleotides and recognized by MutSß. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hydrolysis by Msh2 and Msh3 with mismatch binding to signal for repair.


Asunto(s)
Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN/química , ADN/química , Proteína 2 Homóloga a MutS/química , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga de MutS , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
19.
PLoS One ; 6(9): e24571, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21949728

RESUMEN

Antibodies are assembled by a highly orchestrated series of recombination events during B cell development. One of these events, class switch recombination, is required to produce the IgG, IgE and IgA antibody isotypes characteristic of a secondary immune response. The action of the enzyme activation induced cytidine deaminase is now known to be essential for the initiation of this recombination event. Previous studies have demonstrated that the immunoglobulin switch regions acquire distinct histone modifications prior to recombination. We now present a high resolution analysis of these histone modifications across the IgE switch region prior to the initiation of class switch recombination in primary human B cells and the human CL-01 B cell line. These data show that upon stimulation with IL-4 and an anti-CD40 antibody that mimics T cell help, the nucleosomes of the switch regions are highly modified on histone H3, accumulating acetylation marks and tri-methylation of lysine 4. Distinct peaks of modified histones are found across the switch region, most notably at the 5' splice donor site of the germline (I) exon, which also accumulates AID. These data suggest that acetylation and K4 tri-methylation of histone H3 may represent marks of recombinationally active chromatin and further implicates splicing in the regulation of AID action.


Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Cromatina/genética , Inmunoglobulina E/genética , Región de Cambio de la Inmunoglobulina/genética , Adolescente , Linfocitos B/efectos de los fármacos , Antígenos CD40/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Niño , Preescolar , Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Sitios Genéticos/efectos de los fármacos , Sitios Genéticos/genética , Humanos , Región de Cambio de la Inmunoglobulina/efectos de los fármacos , Interleucina-4/farmacología , Tonsila Palatina/inmunología , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética
20.
Proc Natl Acad Sci U S A ; 104(9): 3078-83, 2007 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-17307873

RESUMEN

The rearrangement of antigen receptor genes is initiated by double-strand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some "cut-and-paste" transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Roturas del ADN de Cadena Simple , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Homeodominio/genética , Mutagénesis , Mutación/genética , Proteínas Nucleares/genética
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