RESUMEN
DNA double-strand breaks (DSBs) are a potentially lethal DNA lesions that disrupt both the physical and genetic continuity of the DNA duplex. Homologous recombination (HR) is a universally conserved genome maintenance pathway that initiates via nucleolytic processing of the broken DNA ends (resection). Eukaryotic DNA resection is catalyzed by the resectosome-a multicomponent molecular machine consisting of the nucleases DNA2 or Exonuclease 1 (EXO1), Bloom's helicase (BLM), the MRE11-RAD50-NBS1 (MRN) complex, and additional regulatory factors. Here, we describe methods for purification and single-molecule imaging and analysis of EXO1, DNA2, and BLM. We also describe how to adapt resection assays to the high-throughput single-molecule DNA curtain assay. By organizing hundreds of individual molecules on the surface of a microfluidic flowcell, DNA curtains visualize protein complexes with the required spatial and temporal resolution to resolve the molecular choreography during critical DNA-processing reactions.
Asunto(s)
Técnicas Analíticas Microfluídicas/métodos , Reparación del ADN por Recombinación , Imagen Individual de Molécula/métodos , Roturas del ADN de Doble Cadena , ADN Helicasas/análisis , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Enzimas Reparadoras del ADN/análisis , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/aislamiento & purificación , Exodesoxirribonucleasas/análisis , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Microscopía Fluorescente/métodos , Puntos Cuánticos/química , RecQ Helicasas/genética , RecQ Helicasas/aislamiento & purificación , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Interstrand DNA cross-links (ICLs) block both replication and transcription, and are commonly repaired by the Fanconi anemia (FA) pathway. However, FA-independent repair mechanisms of ICLs remain poorly understood. Here we report a previously uncharacterized protein, SAN1, as a 5' exonuclease that acts independently of the FA pathway in response to ICLs. Deletion of SAN1 in HeLa cells and mouse embryonic fibroblasts causes sensitivity to ICLs, which is prevented by re-expression of wild type but not nuclease-dead SAN1. SAN1 deletion causes DNA damage and radial chromosome formation following treatment with Mitomycin C, phenocopying defects in the FA pathway. However, SAN1 deletion is not epistatic with FANCD2, a core FA pathway component. Unexpectedly, SAN1 binds to Senataxin (SETX), an RNA/DNA helicase that resolves R-loops. SAN1-SETX binding is increased by ICLs, and is required to prevent cross-link sensitivity. We propose that SAN1 functions with SETX in a pathway necessary for resistance to ICLs.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/fisiología , Exodesoxirribonucleasas/metabolismo , ARN Helicasas/metabolismo , Transactivadores/metabolismo , Animales , ADN Helicasas/metabolismo , Pruebas de Enzimas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Femenino , Fibroblastos , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Noqueados , Enzimas Multifuncionales , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Transactivadores/genética , Transactivadores/aislamiento & purificaciónRESUMEN
Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharacterized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a processive, single-strand DNA-specific exonuclease that degrades DNA in the 5' â 3' direction. TK1252p binds specifically to the GINS15 subunit of T. kodakaraensis GINS complex and this interaction stimulates the exonuclease activity in vitro. This novel archaeal nuclease, designated GINS-associated nuclease (GAN), also forms a complex in vivo with the euryarchaeal-specific DNA polymerase D. Roles for GAN in replisome assembly and DNA replication are discussed.
Asunto(s)
Proteínas Arqueales/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas Arqueales/aislamiento & purificación , Proteínas Cromosómicas no Histona/aislamiento & purificación , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/aislamiento & purificación , Thermococcus/enzimologíaRESUMEN
Artemis is a member of the beta-CASP family of nucleases in the metallo-beta-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His](6)-Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activities, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activities on a Ni-agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease.
Asunto(s)
Biocatálisis , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína Quinasa Activada por ADN/metabolismo , ADN/química , Desoxirribonucleasa I/metabolismo , Exodesoxirribonucleasas/metabolismo , Fraccionamiento Químico , ADN/genética , ADN/metabolismo , Desoxirribonucleasa I/aislamiento & purificación , Exodesoxirribonucleasas/aislamiento & purificación , Células HeLa , HumanosRESUMEN
The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes.
Asunto(s)
Proteínas Arqueales/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Pyrococcus furiosus/metabolismo , Proteínas Arqueales/aislamiento & purificación , ADN/metabolismo , Endodesoxirribonucleasas/aislamiento & purificación , Exodesoxirribonucleasas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Mapeo de Interacción de Proteínas , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genéticaRESUMEN
The first step of homology-dependent DNA double-strand break (DSB) repair is the 5' strand-specific processing of DNA ends to generate 3' single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5' strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5'-->3' degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5'-->3' degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN , ADN de Cadena Simple/metabolismo , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas de Xenopus/metabolismo , Animales , ADN/química , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/aislamiento & purificación , Endonucleasas/antagonistas & inhibidores , Endonucleasas/aislamiento & purificación , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/aislamiento & purificación , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/aislamiento & purificación , Xenopus laevisRESUMEN
We previously demonstrated that a recombinant herpes simplex virus containing a mutation within the finger domain of DNA polymerase replicated DNA with increased fidelity. In this study, we demonstrate that, compared with wild-type polymerase, the mutant enzyme exhibited improved nucleotide selectivity and a reduced ability to extend from mismatched primer termini, which would contribute to the increased DNA replication fidelity.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Mutación , Proteínas Virales/metabolismo , Animales , Disparidad de Par Base , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Humanos , Cinética , Nucleótidos/metabolismo , Estructura Terciaria de Proteína , Spodoptera/citología , Spodoptera/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
NurA is a novel 5'-3' exonuclease that is closely linked to Mre11 and Rad50 homologues in most thermophilic archaea. We report a physical and functional interaction between NurA (StoNurA) and single-stranded DNA-binding protein (StoSSB) from the hyperthermophilic archaeon Sulfolobus tokodaii. StoSSB was identified as a novel StoNurA-interacting protein by pull-down assay using Ni-NTA agarose beads and MALDI-TOF mass spectrometry. The direct interaction between StoNurA and StoSSB was further confirmed by yeast two-hybrid and co-immunoprecipitation analysis. The interaction was supposed to have functional significance because it was found that StoSSB inhibited the 5'-3' ssDNA and dsDNA exonuclease and ssDNA endonuclease activities of StoNurA. Our results suggest that NurA may function closely together with SSB in DNA transactions in archaea.
Asunto(s)
Proteínas Arqueales/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Sulfolobus/metabolismo , Proteínas Arqueales/genética , ADN/química , Reparación del ADN , ADN de Cadena Simple/química , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/aislamiento & purificación , Inmunoprecipitación , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sulfolobus/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The complexes of repair DNA polymerase beta with 3'-exonuclease and some other proteins were isolated from the chromatin of hepatocytes of normal rats for the first time. Biopolymers were extracted from the chromatin by the solution of NaCl and Triton X-100. The extract was fractionated by gel-filtration on Sephacryl S-300 columns successively in low and high ionic strength solutions, on hydroxyapatite, and on Sephadex G-100 columns. The complexes have molecular weights of 100 and 300 kDa. They dissociate to DNA polymerase and exonuclease in the course of chromatography on a DNA-cellulose column or after gel-filtration in the presence of 1 M NaCl. The co-purification of the polymerase and exonuclease is reconstituted in 0.1 M NaCl. The fidelity of monomeric and composite DNA polymerase beta was measured using phage phiX174 amber 3 as a primer/template. The products of the synthesis were transfected into Escherichia coli spheroplasts, and the frequency of reverse mutations was determined. The complex of DNA polymerase beta with 3'-exonuclease was shown to be 30 times more accurate than the monomeric polymerase, which can decrease the probability of repair mutagenesis and carcinogenesis.
Asunto(s)
Bacteriófago phi X 174/química , ADN Polimerasa beta/química , ADN Viral/química , Exodesoxirribonucleasas/química , Hepatocitos/enzimología , Animales , Bacteriófago phi X 174/genética , ADN Polimerasa beta/aislamiento & purificación , ADN Viral/genética , Escherichia coli/genética , Exodesoxirribonucleasas/aislamiento & purificación , Mutación , Ratas , Esferoplastos/genéticaRESUMEN
The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 5'-end, liberating mono-, di- and polynucleotides terminating with a 5' phosphate. Exonuclease IX is a paralogue of the small fragment of Escherichia coli DNA polymerase I, a FEN with which it shares 66% similarity. Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB. Immobilized ExoIX was able to recover SSB from E. coli lysates both in the presence and absence of DNA. In vitro cross-linking studies carried out in the absence of DNA showed that the SSB tetramer appears to bind up to two molecules of ExoIX. Furthermore, we found that a 3'-5' exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography. The associated 3'-5' exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Exodesoxirribonucleasas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Reactivos de Enlaces Cruzados , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/aislamiento & purificaciónRESUMEN
Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.
Asunto(s)
ADN/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/aislamiento & purificación , Streptomyces coelicolor/enzimología , Secuencia de Aminoácidos , Bacteriófago lambda/genética , Secuencia de Bases , Tampones (Química) , Cationes Bivalentes/química , Clonación Molecular , AMP Cíclico/metabolismo , ADN/química , ADN de Cadena Simple/metabolismo , ADN Superhelicoidal/metabolismo , ADN Viral/metabolismo , Exodesoxirribonucleasas/biosíntesis , Exodesoxirribonucleasas/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.
Asunto(s)
Disparidad de Par Base , Exodesoxirribonucleasas/metabolismo , Sitios de Unión , ADN/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Desoxirribonucleótidos/química , Desoxirribonucleótidos/metabolismo , Endonucleasas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Humanos , Enzimas Multifuncionales , Mutación , Hidrolasas Diéster Fosfóricas/metabolismo , Especificidad por SustratoRESUMEN
Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5' flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known about the role of human DNA2 (hDNA2), although complementation experiments have shown that it can function in yeast to replace ScDNA2. We have now characterized the biochemical properties of hDna2. Recombinant hDna2 has single-stranded DNA-dependent ATPase and DNA helicase activity. It also has 5'-3' nuclease activity with preference for single-stranded 5' flaps adjacent to a duplex DNA region. The nuclease activity is stimulated by RPA and suppressed by steric hindrance at the 5' end. Moreover, hDna2 shows strong 3'-5' nuclease activity. This activity cleaves single-stranded DNA in a fork structure and, like the 5'-3' activity, is suppressed by steric hindrance at the 3'-end, suggesting that the 3'-5' nuclease requires a 3' single-stranded end for activation. These biochemical specificities are very similar to those of the ScDna2 protein, but suggest that the 3'-5' nuclease activity may be more important than previously thought.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Desoxirribonucleasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Animales , Baculoviridae/genética , Línea Celular , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , ADN de Cadena Simple/metabolismo , Desoxirribonucleasas/genética , Desoxirribonucleasas/aislamiento & purificación , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/aislamiento & purificación , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Exodesoxirribonucleasas/metabolismo , Células HeLa , Humanos , Insectos/citología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2' DNA and the resulting protein-DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn2+-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Guanina/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/química , ADN/química , Replicación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Endodesoxirribonucleasas/aislamiento & purificación , Exodesoxirribonucleasas/aislamiento & purificación , G-Cuádruplex , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Especificidad por Sustrato , Telómero/metabolismoRESUMEN
Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Exodesoxirribonucleasas/metabolismo , Thermotoga maritima/enzimología , Cationes Bivalentes/farmacología , Clonación Molecular , Cartilla de ADN , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/aislamiento & purificación , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Cinética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
With the aim to identify unconventional DNA polymerases from human cells, we have set up a special assay to fractionate HeLa extracts based on the ability (i) to bypass DNA lesions, (ii) to be resistant to aphidicolin and an inhibitory antibody against pol alpha and (iii) to be non-responsive to proliferating cell nuclear antigen. After eight different chromatographic steps, an aphidicolin-resistant DNA polymerase activity was obtained that was able to utilize either undamaged or abasic sites-containing DNA with the same efficiency. Biochemical characterization and immunoblot analysis allowed its identification as the human homologue of DNA polymerase theta (hpol theta), whose cDNA has been cloned by homology with the mus308 gene of Drosophila melanogaster but still awaited detailed biochemical characterization. The purified hpol theta was devoid of detectable helicase activity, possessed a 3'-->5' exonuclease activity and showed biochemical properties clearly distinct from any other eukaryotic DNA polymerase known so far. Misincorporation and fidelity assays showed that: (i) hpol theta was able to catalyze efficiently DNA synthesis past an abasic site; and (ii) hpol theta showed high fidelity. Our findings are discussed in light of the proposed physiological role of hpol theta.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Mutagénesis/genética , ADN/biosíntesis , ADN/genética , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Exodesoxirribonucleasas/metabolismo , Células HeLa , Humanos , Cinética , Moldes Genéticos , Termodinámica , ADN Polimerasa thetaRESUMEN
We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.
Asunto(s)
Disparidad de Par Base , Reparación del ADN , Exodesoxirribonucleasas/metabolismo , Línea Celular , Enzimas Reparadoras del ADN , Exodesoxirribonucleasas/aislamiento & purificación , Humanos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coli RecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60 degrees C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5'-->3' exonuclease activity, which was enhanced by increasing the temperature to 50 degrees C. Mg(2+), Mn(2+) or Co(2+) ions were required to activate both proteins, whereas Ca(2+) and Zn(2+) had no effects.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Exodesoxirribonucleasas/genética , Thermus thermophilus/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Exodesoxirribonucleasas/aislamiento & purificación , Exodesoxirribonucleasas/metabolismo , Exonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Genoma Bacteriano , Cinética , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
The RecB subunit of the Escherichia coli RecBCD enzyme has both helicase and nuclease activities. The helicase function was localized to an N-terminal domain, whereas the nuclease activity was found in a C-terminal domain. Recent analysis has uncovered a group of proteins that have weak amino acid sequence similarity to the RecB nuclease domain and that are proposed to constitute a family of related proteins (Aravind, L., Walker, D. R., and Koonin, E. V. (1999) Nucleic Acids Res. 27, 1223-1242). One is the E. coli RecE protein (exonuclease VIII), an ATP-independent exonuclease that degrades the 5'-terminated strand of double-stranded DNA. We have made mutations in several residues of RecE that align with the critical residues of RecB, and we find that the mutations reduce or abolish the nuclease activity of RecE but do not affect the enzyme binding to linear double-stranded DNA. Proteolysis experiments with subtilisin show that a stable 34-kilodalton C-terminal domain that contains these critical residues has nuclease activity, whereas no stable proteolytic fragments accumulate from the N-terminal portion of RecE. These results show that RecE has a nuclease domain and active site that are similar to RecB, despite the very weak sequence similarity between the two proteins. These similarities support the hypothesis that the nuclease domains of the two proteins are evolutionarily related.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Exodesoxirribonucleasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase activity is associated with IgGs from the sera of patients with multiple sclerosis (MS) but not with those from the sera of normal humans. Here we present evidence showing that MS IgG, its F(ab) fragments, and separated L-chains catalyze DNA hydrolysis. The properties of the DNase activity of these polyclonal IgGs distinguish them from other known human DNases. In addition, their specific activities with different oligonucleotide substrates and the range of optimal pHs, apparent K(M) values and substrate specificities varied widely for different patients. The findings speak in favor of the generation by the immune systems of individual patients of a variety of polyclonal catalytic IgG pools, from relatively small to extremely large ones.