RESUMEN

Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A small middle dotT base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a "synaptic pathway" involving a three-stranded nucleoprotein intermediate, rather than by a "helicase pathway" involving the separation and reannealing of DNA strands.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Meiosis/fisiología , Recombinación Genética , Adenosina Trifosfatasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Desoxirribonucleasas/metabolismo , Humanos , Masculino , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

The bacterial RecA protein has been the most intensively studied enzyme in homologous genetic recombination. The core of RecA is structurally homologous to that of the F1-ATPase and helicases. Like the F1-ATPase and ring helicases, RecA forms a hexameric ring. The human Dmc1 (hDmc1) protein, a meiosis-specific recombinase, is homologous to RecA. We show that hDmc1 forms octameric rings. Unlike RecA and Rad51, however, hDmc1 protein does not form helical filaments. The hDmc1 ring binds DNA in the central channel, as do the ring helicases, which is likely to represent the active form of the protein. These observations indicate that the conservation of the RecA-like ring structure extends from bacteria to humans, and that some RecA homologs may form both rings and filaments, whereas others may function only as rings.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Bacteriófagos/metabolismo , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Humanos , Microscopía Electrónica , Conformación Proteica , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo

RESUMEN

The beta protein of bacteriophage lambda acts in homologous genetic recombination by catalyzing the annealing of complementary single-stranded DNA produced by the lambda exonuclease. It has been shown that the beta protein binds to the products of the annealing reaction more tightly than to the initial substrates. We find that beta protein exists in three structural states. In the absence of DNA, beta protein forms inactive rings with approximately 12 subunits. The active form of the beta protein in the presence of oligonucleotides or single-stranded DNA is a ring, composed of approximately 15-18 subunits. The double-stranded products of the annealing reaction catalyzed by the rings are bound by beta protein in a left-handed helical structure, which protects the products from nucleolytic degradation. These observations suggest structural homology for a family of proteins, including the phage P22 erf, the bacterial RecT, and the eukaryotic Rad52 proteins, all of which are involved in homologous recombination.

Asunto(s)

Bacteriófago lambda/química , Proteínas de Unión al ADN/ultraestructura , Proteínas Virales/ultraestructura , Bacteriófago lambda/metabolismo , ADN/química , ADN/metabolismo , ADN/ultraestructura , ADN Viral/química , ADN Viral/metabolismo , ADN Viral/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestructura , Proteínas Virales/química , Proteínas Virales/metabolismo

RESUMEN

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after gamma irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Micronúcleos con Defecto Cromosómico/metabolismo , Células 3T3 , Animales , Ciclo Celular , Línea Celular Transformada , Núcleo Celular , Daño del ADN , Reparación del ADN , Humanos , Ratones , Mutágenos , Recombinasa Rad51 , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Replicación A

RESUMEN

The eukaryotic homologs of RecA protein are central enzymes of recombination and repair, and notwithstanding a high degree of conservation they differ sufficiently from RecA to offer insights into mechanisms and biological roles. The yield of DNA strand exchange reactions driven by both Escherichia coli RecA protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC composition, HsRad51 promoted less exchange than RecA. When 40% of bases were GC pairs, the rate constant for strand exchange by HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA. The ability of HsRad51 to form joints in the absence of net strand exchange was confirmed by experiments in which heterologous blocks at both ends of linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization. These findings suggest that HsRad51 acting alone on human DNA in vivo is a pairing protein that cannot form extensive heteroduplex DNA.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Conformación Proteica , Disparidad de Par Base , Catálisis , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Humanos , Cloruro de Magnesio/farmacología , Peso Molecular , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Recombinasa Rad51 , Rec A Recombinasas/metabolismo , Recombinación Genética

RESUMEN

Human Rad51 belongs to a ubiquitous family of proteins that enable a single strand to recognize homology in duplex DNA, and thereby to initiate genetic exchanges and DNA repair, but the mechanism of recognition remains unknown. Kinetic analysis by fluorescence resonance energy transfer combined with the study of base substitutions and base mismatches reveals that recognition of homology, helix destabilization, exchange of base pairs, and initiation of strand exchange are integral parts of a rapid, concerted mechanism in which A:T base pairs play a critical role. Exchange of base pairs is essential for recognition of homology, and physical evidence indicates that such an exchange occurs early enough to mediate recognition.

Asunto(s)

Adenina/metabolismo , Emparejamiento Base/genética , Intercambio Genético/genética , Proteínas de Unión al ADN/metabolismo , ADN/genética , Homología de Secuencia de Ácido Nucleico , Timina/metabolismo , Disparidad de Par Base/genética , Secuencia de Bases , Citosina/metabolismo , ADN/química , ADN/metabolismo , Reparación del ADN/genética , Fluorescencia , Guanina/metabolismo , Humanos , Inosina/genética , Inosina/metabolismo , Cinética , Mutación/genética , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Recombinasa Rad51

RESUMEN

Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.

Asunto(s)

ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinación Genética , Animales , Células Cultivadas , Proteínas de Unión al ADN/aislamiento & purificación , Fibroblastos/metabolismo , Rayos gamma , Humanos , Ratones , Peso Molecular , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Pruebas de Precipitina , Recombinasa Rad51 , Ratas , Proteína de Replicación A

RESUMEN

Homologs of Escherichia coli RecA recombination protein, which have been found throughout the living kingdom, promote homologous pairing and strand exchange. The nucleoprotein filament, within which strand exchange occurs, has been conserved through evolution, but conservation of the polarity of exchange and the significance of that directionality has not been settled. Using oligonucleotides as substrates, and assays based on fluorescence resonance energy transfer (FRET), we distinguished the biased formation of homologous joints at either end of duplex DNA from the subsequent directionality of strand exchange. As with E. coli RecA protein, the homologous Rad51 proteins from both Homo sapiens (HsRad51) and Saccharomyces cerevisiae (ScRad51) propagated DNA strand exchange preferentially in the 5' to 3' direction. The data suggest that 5' to 3' polarity is a conserved intrinsic property of recombination filaments.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , ADN/genética , ADN/metabolismo , Proteínas Fúngicas/metabolismo , Rec A Recombinasas/metabolismo , Recombinación Genética , Composición de Base , Secuencia de Bases , ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Modelos Biológicos , Nucleoproteínas/metabolismo , Recombinasa Rad51 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae

RESUMEN

Bacteriophage lambda encodes a 28 kDa protein called beta that binds to single-stranded DNA and promotes the renaturation of complementary single strands. beta Protein fails to bind directly to duplex DNA but remains bound to the DNA product of renaturation that beta itself catalyzes. These observations led to an examination of the ability of beta protein to promote strand exchange. beta Protein caused the replacement of a 43-mer oligonucleotide annealed to M13 circular single-stranded DNA by a homologous 63-mer whose 20 extra nucleotide residues were complementary to the adjacent 3' region of M13 DNA. The role of beta protein in this reaction was manifested in several ways: beta protein pushed the exchange through four to eight mismatches, which blocked exchange mediated by spontaneous renaturation and branch migration; beta imposed a polarity on the strand exchange that was lacking in the spontaneous reaction; and beta remained bound to the heteroduplex product of strand exchange. These observations reveal a mechanism by which a protein can drive strand exchange in one direction without using ATP or any other exogenous source of energy.

Asunto(s)

Bacteriófago lambda/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Bacteriófago M13/genética , Bacteriófago lambda/genética , Secuencia de Bases , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN Viral/química , ADN Viral/genética , Sustancias Macromoleculares , Modelos Biológicos , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Renaturación de Ácido Nucleico , Unión Proteica , Recombinación Genética

RESUMEN

Phage lambda encodes two recombination proteins that are required for homologous recombination in a recA- host strain. Of these two recombination proteins, one is an exonuclease whose action on double-stranded DNA produces 3' single-stranded ends; the other, called beta protein, is a DNA binding protein that promotes the renaturation of complementary single strands. The enzymes of phage lambda provide a model for understanding a recombination pathway called "single-strand annealing". Further investigation of the binding of beta protein to DNA has revealed a new mechanism of renaturation. As reported before, beta protein binds directly to single-stranded DNA, but not to double-stranded DNA. However, in the experiments reported here, we observed that beta protein bound more strongly to a presumed intermediate in the renaturation reaction that beta itself catalyzed, and beta thereby protected all of a renatured duplex 83-mer oligonucleotide from nuclease digestion.

Asunto(s)

Bacteriófago lambda/metabolismo , ADN Viral/química , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Bacteriófago lambda/genética , Sitios de Unión , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN Viral/genética , Proteínas de Unión al ADN/química , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares , Datos de Secuencia Molecular , Renaturación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Recombinación Genética , Proteínas Virales/química

RESUMEN

The cDNA for human protein HsRad54, which is a structural homolog of Saccharomyces cerevisiae recombination/repair protein Rad54, was cloned and expressed in Escherichia coli. As demonstrated by analysis in vitro and in vivo, HsRad54 protein interacts with human Rad51 recombinase. The interaction is mediated by the N-terminal domain of HsRad54 protein, which interacts with both free and DNA-bound HsRad51 protein.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Western Blotting , Clonación Molecular , ADN/metabolismo , ADN Helicasas , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN/genética , Interacciones Farmacológicas , Escherichia coli/genética , Proteínas Fúngicas/genética , Expresión Génica , Humanos , Fragmentos de Péptidos/metabolismo , Recombinasa Rad51 , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae

RESUMEN

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases. Oligonucleotide sequences that produced 2-14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences. The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%. These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange.

Asunto(s)

Rec A Recombinasas/metabolismo , Recombinación Genética , Composición de Base , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Análisis de Inyección de Flujo , Ácidos Nucleicos Heterodúplex/metabolismo , Espectrometría de Fluorescencia/métodos

RESUMEN

Meiosis-specific homologs of RecA protein have been identified in Saccharomyces cerevisiae and higher eukaryotes including mammals, but their enzymatic activities have not been described. We have purified the human protein HsDmc1 produced in Escherichia coli from a cloned copy of the cDNA. The recombinant enzyme had DNA-dependent ATPase activity with an estimated kcat of 1.5 min-1. DNase protection experiments with oligonucleotides as substrates indicated that HsDmc1 protein binds preferentially to single-stranded DNA with a stoichiometry of approximately one molecule of protein per three nucleotide residues. HsDmc1 protein catalyzed the formation of D-loops in superhelical DNA, as well as strand exchange between single-stranded and double-stranded oligonucleotides. The requirements for strand exchange catalyzed by HsDmc1 were similar to those of RecA protein, but exchange caused by HsDmc1 was not supported by ATPgammaS.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Rec A Recombinasas/metabolismo , Recombinación Genética , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN de Cadena Simple/metabolismo , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I , Escherichia coli , Humanos , Meiosis , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae

RESUMEN

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. In this study, we measured RecA-catalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes on the ends of deoxyribo-oligonucleotides. By varying the position of the dyes in separate assays, we were able to detect the pairing of single-stranded RecA filament with duplex DNA as an increase in energy transfer, and strand displacement as a decrease in energy transfer. With these assays, the kinetics of pairing and strand displacement were studied by stopped-flow spectrofluorometry. The data revealed a rapid, second order, reversible pairing step that was followed by a slower, reversible, first order strand exchange step. These data indicate that an initial unstable intermediate exists which can readily return to reactants, and that a further, rate-limiting step (or steps) is required to effect or complete strand exchange.

Asunto(s)

ADN de Cadena Simple/metabolismo , Escherichia coli/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Rec A Recombinasas/metabolismo , Secuencia de Bases , Catálisis , Transferencia de Energía , Colorantes Fluorescentes , Cinética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Rodaminas , Espectrometría de Fluorescencia , Especificidad por Sustrato

RESUMEN

The nucleoprotein filament formed on a circular single strand by Escherichia coli RecA protein in vitro can pair with homologous duplex DNA even when the latter lacks a free homologous end, but subsequent progression of the reaction through strand exchange requires an end in at least one strand of the duplex DNA. We purified from E. coli an endonuclease activity that cleaves the outgoing strand of duplex DNA at the junction of homologous and heterologous sequences in three-stranded RecA-recombination intermediates. This endonuclease activity also cleaves specifically at the junctions of duplex and single-stranded regions in synthetic double-stranded oligonucleotides whose central portion consists of unpaired heterologous sequences. These activities are consistent with a role in recombination and repair of DNA.

Asunto(s)

Endonucleasas/metabolismo , Escherichia coli/metabolismo , Rec A Recombinasas/metabolismo , Recombinación Genética , Secuencia de Bases , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Reparación del ADN , Electroforesis en Gel de Poliacrilamida , Endonucleasas/aislamiento & purificación , Escherichia coli/genética , Genotipo , Modelos Estructurales , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Especificidad por Sustrato

RESUMEN

In the yeast, Saccharomyces cerevisiae, the Rad52 gene is important for both mitotic and meiotic recombination. Homologs of the Rad52 gene have been identified in several eukaryotic organisms, ranging from yeast to man. As reported here, human Rad52 protein binds to both single- and double-stranded DNA; and acting on a pair of single-stranded and partially duplex substrates it promotes annealing of complementary strands of DNA, which is followed by branch migration.

Asunto(s)

Reparación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Clonación Molecular , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Agar , Escherichia coli/genética , Humanos , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Homologous pairing and strand exchange, which are catalyzed by Escherichia coli RecA protein, are central to homologous recombination. Homologs of this protein are found in eukaryotes; however, little has been reported on the recombinase activities of the mammalian homologs, including the human protein, denoted HsRad51. For the studies described here, we purified HsRad51 form E. coli. Although the activities of HsRad51 and RecA were qualitatively similar in the presence of ATP, there were also striking differences. The stoichiometry of binding to DNA and the rate of renaturation of complementary strands were similar for the two proteins, but rates of ATP hydrolysis, homologous pairing, and subsequent strand exchange promoted by HsRad51 were less than 1/10 those of RecA. In addition, HsRad51 bound gamma-thio-ATP and formed stable presynaptic complexes that promoted renaturation as rapidly as RecA, but the recombinant human protein catalyzed neither strand exchange nor homologous pairing of a single strand with duplex DNA in the presence of the ATP analog. By contrast, RecA promoted both of the latter reactions in control experiments. These observations suggest that among RecA-like proteins, HsRad51 may be a variant in which homologous pairing and strand exchange are more closely linked to the hydrolysis of ATP.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Recombinación Genética , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Desoxirribonucleasa I/metabolismo , Transferencia de Energía , Humanos , Datos de Secuencia Molecular , Renaturación de Ácido Nucleico , Oligonucleótidos/química , Unión Proteica , Recombinasa Rad51 , Espectrometría de Fluorescencia

RESUMEN

Using the yeast two-hybrid system, we isolated a cDNA encoding a novel human protein, named Pir51, that strongly interacts with human Rad51 recombinase. Analysis in vitro confirmed the interaction between Rad51 and Pir51. Pir51 mRNA is expressed in a number of human organs, most notably in testis, thymus, colon and small intestine. The Pir51 gene locus was mapped to chromosome 12p13.1-13. 2 by fluorescence in situ hybridization. The Pir51 protein was expressed in Escherichia coli and purified to near homogeneity. Biochemical analysis shows that the Pir51 protein binds both single- and double-stranded DNA, and is capable of aggregating DNA. The protein also binds RNA. The Pir51 protein may represent a new member of the multiprotein complexes postulated to carry out homologous recombination and DNA repair in mammalian cells.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Genes , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Humanos Par 12/genética , ADN/metabolismo , ADN Complementario/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Especificidad de Órganos , Unión Proteica , ARN/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de Unión al ARN , Recombinasa Rad51 , Recombinación Genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae

RESUMEN

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270-328 (from the N terminus). Our model building pointed out the possibility that this domain is a part of "gateway" through which double-stranded DNA finds a path for direct contact with single-stranded DNA within a presynaptic RecA filament in the search for homology. To test this possible function of the domain, we made mutant RecA proteins by site-directed single (or double, in one case) replacement of 2 conserved basic amino acid residues and 5 among 9 nonconserved basic amino acid residues in the domain. Replacement of either of the 2 conserved amino acid residues caused deficiencies in repair of UV-damaged DNA, an in vivo function of RecA protein, whereas the replacement of most (except one) of the tested nonconserved ones gave little or no effect. Purified mutant RecA proteins showed no (or only slight) deficiencies in the formation of presynaptic filaments as assessed by various assays. However, presynaptic filaments of both proteins that had replacement of a conserved amino acid residue had significant defects in binding to and pairing with duplex DNA (secondary binding). These results are consistent with our model that the conserved amino acid residues in the C-terminal domain have a direct role in double-stranded DNA binding and that they constitute a part of a gateway for homologous recognition.

Asunto(s)

ADN/metabolismo , Rec A Recombinasas/química , Adenosina Trifosfato/metabolismo , Arginina , Sitios de Unión , Lisina , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica

RESUMEN

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

Asunto(s)

Linfocitos B/inmunología , Proteínas de Unión al ADN/biosíntesis , Cambio de Clase de Inmunoglobulina , Lipopolisacáridos/farmacología , Recombinación Genética , Bazo/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos , Linfocitos B/efectos de los fármacos , Células Cultivadas , Mapeo Cromosómico , Secuencia Conservada , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/inmunología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes de Inmunoglobulinas , Humanos , Inmunización , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Inmunohistoquímica , Cinética , Ratones , Ratones Endogámicos BALB C , Recombinasa Rad51 , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Factores de Tiempo