RESUMEN

Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.

Asunto(s)

Reparación del ADN/fisiología , Proteínas de Unión al ADN/aislamiento & purificación , Testículo/química , Adenosina Trifosfatasas/metabolismo , Baculoviridae/genética , Cromatografía en Gel , Reparación del ADN/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Microscopía Electrónica , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Recombinasa Rad51 , Proteínas Recombinantes/metabolismo , Recombinación Genética , Testículo/citología

RESUMEN

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60-70 degrees C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.

Asunto(s)

Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleoproteínas/metabolismo , Recombinación Genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Archaeoglobus fulgidus/química , ADN/química , ADN/metabolismo , ADN/ultraestructura , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Microscopía Electrónica , Conformación de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/ultraestructura , Unión Proteica , Conformación Proteica , Temperatura

RESUMEN

Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

Asunto(s)

Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Recombinación Genética , Animales , Baculoviridae/metabolismo , Línea Celular , ADN Complementario/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Insectos , Microscopía Electrónica , Modelos Genéticos , Plásmidos/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52 , Proteínas Recombinantes/metabolismo , Factores de Tiempo

RESUMEN

In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C--XRCC3 complex binds single-stranded, but not duplex DNA, to form protein--DNA networks that have been visualized by electron microscopy.

Asunto(s)

Reparación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , Humanos , Ratones , Microscopía Electrónica , Oligodesoxirribonucleótidos/metabolismo , Conejos , Recombinasa Rad51 , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Spodoptera

RESUMEN

Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.

Asunto(s)

Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/metabolismo , Recombinación Genética , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Proteína BRCA2 , Sitios de Unión , Neoplasias de la Mama/genética , Cromatografía en Gel , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Humanos , Microscopía Electrónica , Modelos Biológicos , Datos de Secuencia Molecular , Peso Molecular , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Nucleoproteínas/antagonistas & inhibidores , Nucleoproteínas/metabolismo , Nucleoproteínas/ultraestructura , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Recombinasa Rad51 , Fracciones Subcelulares , Especificidad por Sustrato , Factores de Transcripción/química , Factores de Transcripción/genética

RESUMEN

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.

Asunto(s)

Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Nucleoproteínas/metabolismo , Rec A Recombinasas/metabolismo , Daño del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/ultraestructura , Proteínas de Escherichia coli , Modelos Genéticos , Nucleoproteínas/ultraestructura

RESUMEN

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

Asunto(s)

Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN/genética , ADN/metabolismo , ADN/química , ADN/ultraestructura , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/ultraestructura , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , ADN Superhelicoidal/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Humanos , Microscopía Electrónica , Modelos Genéticos , Conformación de Ácido Nucleico , Unión Proteica , Recombinasa Rad51 , Rec A Recombinasas/metabolismo , Recombinación Genética/genética , Proteína de Replicación A , Homología de Secuencia de Ácido Nucleico

RESUMEN

The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation [1]. Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways [2] [3] [4]. Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing [5] [6] [7] and to stimulate Rad51-mediated homologous pairing [8] [9] [10] [11]. Electron microscopic examinations of the yeast [12] and human [13] Rad52 proteins have revealed their assembly into ring-like structures in vitro. Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings. A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel. Like the hexameric helicases such as Escherichia coli DnaB [14] [15], bacteriophage T7 gp4b [16] [17], simian virus 40 (SV40) large T antigen [18] and papilloma virus E1 [19], the Rad52 rings show a distinctly chiral arrangement of subunits. Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP.

Asunto(s)

Proteínas de Unión al ADN/ultraestructura , Animales , Línea Celular , Humanos , Proteína Recombinante y Reparadora de ADN Rad52 , Proteínas Recombinantes de Fusión/ultraestructura

RESUMEN

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Proteínas de Ciclo Celular , ADN Nucleotidiltransferasas/metabolismo , ADN Nucleotidiltransferasas/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Integrasas , Adenosina Trifosfatasas/aislamiento & purificación , Clonación Molecular , ADN Nucleotidiltransferasas/aislamiento & purificación , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/química , ADN Viral/biosíntesis , ADN Viral/química , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/genética , Biblioteca de Genes , Humanos , Masculino , Meiosis , Microscopía Electrónica , Ácidos Nucleicos Heterodúplex/biosíntesis , Ácidos Nucleicos Heterodúplex/química , Especificidad de Órganos , Recombinasa Rad51 , Rec A Recombinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Recombinasas , Recombinación Genética , Testículo/enzimología

RESUMEN

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

Asunto(s)

Antígenos Nucleares , ADN Helicasas , Reparación del ADN/genética , Proteínas de Unión al ADN , ADN/genética , Proteínas de Saccharomyces cerevisiae , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Humanos , Autoantígeno Ku , Modelos Genéticos , Proteínas Nucleares/genética , Recombinación Genética , Homología de Secuencia

RESUMEN

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

Asunto(s)

Daño del ADN , ADN/química , Cloruro de Magnesio/farmacología , Conformación de Ácido Nucleico , Cloruro de Sodio/farmacología , ADN/metabolismo , Congelación , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , Modelos Moleculares , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo

RESUMEN

Axial deflection of DNA molecules in solution results from thermal motion and intrinsic curvature related to the DNA sequence. In order to measure directly the contribution of thermal motion we constructed intrinsically straight DNA molecules and measured their persistence length by cryo-electron microscopy. The persistence length of such intrinsically straight DNA molecules suspended in thin layers of cryo-vitrified solutions is about 80 nm. In order to test our experimental approach, we measured the apparent persistence length of DNA molecules with natural "random" sequences. The result of about 45 nm is consistent with the generally accepted value of the apparent persistence length of natural DNA sequences. By comparing the apparent persistence length to intrinsically straight DNA with that of natural DNA, it is possible to determine both the dynamic and the static contributions to the apparent persistence length.

Asunto(s)

ADN/química , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Electrónica/métodos , Modelos Moleculares , Conformación de Ácido Nucleico , Secuencia de Bases , Congelación , Modelos Químicos , Datos de Secuencia Molecular , Método de Montecarlo

Asunto(s)

ADN/química , Modelos Moleculares , Conformación de Ácido Nucleico , Secuencia de Bases , Criopreservación , ADN/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular

RESUMEN

Paul Howard-Flanders et al proposed a molecular model of RecA-mediated recombination reaction six years ago. How does this model stand at present? In answering this question, we focus on two leading ideas of the original model, namely the proposal of the coaxial arrangement of the aligned DNA molecules within helical RecA filaments and the proposal of the ATP independence of the pairing stage of the recombination reaction. Results obtained after the model was proposed are reviewed and compared with these original assumptions and postulates of the model. EM visualization of recombining DNA molecules, studies of the energetics of the RecA-mediated recombination reaction and biochemical analysis of deproteinized joint molecules are fully consistent with a triple-stranded DNA arrangement during the RecA-mediated recombination reaction and demonstrate the ATP independence of the pairing stage of the reaction.

Asunto(s)

ADN/metabolismo , Rec A Recombinasas/metabolismo , Recombinación Genética , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , ADN de Cadena Simple/metabolismo , Microscopía Electrónica , Modelos Biológicos , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo

RESUMEN

The shape of supercoiled DNA molecules in solution is directly visualized by cryo-electron microscopy of vitrified samples. We observe that: (i) supercoiled DNA molecules in solution adopt an interwound rather than a toroidal form, (ii) the diameter of the interwound superhelix changes from about 12 nm to 4 nm upon addition of magnesium salt to the solution and (iii) the partition of the linking deficit between twist and writhe can be quantitatively determined for individual molecules.

Asunto(s)

ADN Superhelicoidal/ultraestructura , Cloruro de Magnesio/farmacología , Conformación de Ácido Nucleico , Soluciones , Estereoisomerismo

Asunto(s)

ADN Bacteriano/metabolismo , Escherichia coli/genética , Rec A Recombinasas/metabolismo , Recombinación Genética , ADN Bacteriano/genética , Escherichia coli/metabolismo , Rec A Recombinasas/genética

Asunto(s)

Bacteriófago lambda/genética , ADN Viral/genética , Recombinación Genética , Sitios de Unión , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , ADN Viral/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transposasas