RESUMEN
DNA lesions such as crosslinks represent obstacles for the replication machinery. Nonetheless, replication can proceed via the DNA damage tolerance pathway also known as postreplicative repair pathway. SNF2 ATPase Rad5 homologs, such as RAD5A of the model plant Arabidopsis thaliana, are important for the error-free mode of this pathway. We able to demonstrate before, that RAD5A is a key factor in the repair of DNA crosslinks in Arabidopsis. Here, we show by in vitro analysis that AtRAD5A protein is a DNA translocase able to catalyse fork regression. Interestingly, replication forks with a gap in the leading strand are processed best, in line with its suggested function. Furthermore AtRAD5A catalyses branch migration of a Holliday junction and is furthermore not impaired by the DNA binding of a model protein, which is indicative of its ability to displace other proteins. Rad5 homologs possess HIRAN (Hip116, Rad5; N-terminal) domains. By biochemical analysis we were able to demonstrate that the HIRAN domain variant from Arabidopsis RAD5A mediates structure selective DNA binding without the necessity for a free 3'OH group as has been shown to be required for binding of HIRAN domains in a mammalian RAD5 homolog. The biological importance of the HIRAN domain in AtRAD5A is demonstrated by our result that it is required for its function in DNA crosslink repair in vivo.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ADN/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN/química , Daño del ADN/genética , Daño del ADN/fisiología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Unión ProteicaRESUMEN
Kissing-loop annealing of nucleic acids occurs in nature in several viruses and in prokaryotic replication, among other circumstances. Nucleobases of two nucleic acid strands (loops) interact with each other, although the two strands cannot wrap around each other completely because of the adjacent double-stranded regions (stems). In this study, we exploited DNA kissing-loop interaction for nanotechnological application. We functionalized the vertices of DNA tetrahedrons with DNA stem-loop sequences. The complementary loop sequence design allowed the hybridization of different tetrahedrons via kissing-loop interaction, which might be further exploited for nanotechnology applications like cargo transport and logical elements. Importantly, we were able to manipulate the stability of those kissing-loop complexes based on the choice and concentration of cations, the temperature and the number of complementary loops per tetrahedron either at the same or at different vertices. Moreover, variations in loop sequences allowed the characterization of necessary sequences within the loop as well as additional stability control of the kissing complexes. Therefore, the properties of the presented nanostructures make them an important tool for DNA nanotechnology.
Asunto(s)
ADN/química , Nanoestructuras/química , Nanotecnología , ADN/genética , Secuencias Invertidas Repetidas , Conformación de Ácido Nucleico , Hibridación de Ácido NucleicoRESUMEN
Helicases are essential for DNA metabolism. Different helicases have different properties tailored to fulfill their specific tasks. RecQ-helicases are known to be important in DNA repair and DNA recombination. In higher organisms several RecQ homologues can be identified. For instance, seven RecQ homologues were identified in the model plant Arabidopsis thaliana. Specialization of those proteins can possibly be reflected by differences in their biochemical substrate spectrum. Moreover, a helicase of interest might be defined by its biochemical properties as a functional ortholog of a RecQ helicase in other organisms. In this chapter the initial steps that will provide the basis for a proper biochemical characterization are given. After the description of the expression of the helicase of interest in the heterologous host Escherichia coli, its purification with the help of two affinity tags and the preparation of a model DNA substrate for the strand displacement assay are described. Finally, it is shown how this model substrate can be used to ensure the purity of the enzymatic preparation of interest.
Asunto(s)
Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , RecQ Helicasas , Proteínas de Arabidopsis/genética , Calmodulina/metabolismo , Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , ADN/química , ADN/genética , ADN/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/aislamiento & purificación , RecQ Helicasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Genetic and biochemical analyses of SRS2 homologs in fungi indicate a function in the processing of homologous recombination (HR) intermediates. To date, no SRS2 homologs have been described and analyzed in higher eukaryotes. Here, we report the first biochemical characterization of an SRS2 homolog from a multicellular eukaryote, the plant Arabidopsis thaliana. We studied the basic properties of AtSRS2 and were able to show that it is a functional 3'- to 5'-helicase. Furthermore, we characterized its biochemical function on recombinogenic intermediates and were able to show the unwinding of nicked Holliday junctions (HJs) and partial HJs (PX junctions). For the first time, we demonstrated strand annealing activity for an SRS2 homolog and characterized its strand pairing activity in detail. Our results indicate that AtSRS2 has properties that enable it to be involved in different steps during the processing of recombination intermediates. On the one hand, it could be involved in the unwinding of an elongating invading strand from a donor strand, while on the other hand, it could be involved in the annealing of the elongated strand at a later step.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Recombinación Genética , Nucleótidos de Adenina/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , ADN Helicasas/química , ADN Cruciforme/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Members of the conserved RecQ helicase family are important for the preservation of genomic stability. Multiple RecQ homologs within one organism raise the question of functional specialization. Whereas five different homologs are present in humans, the model plant Arabidopsis (Arabidopsis thaliana) carries seven RecQ homologs in its genome. We performed biochemical analysis of AtRECQ3, expanded upon a previous analysis of AtRECQ2, and compared their properties. Both proteins differ in their domain composition. Our analysis demonstrates that they are 3' to 5' helicases with similar activities on partial duplex DNA. However, they promote different outcomes with synthetic DNA structures that mimic Holliday junctions or a replication fork. AtRECQ2 catalyzes Holliday junction branch migration and replication fork regression, while AtRECQ3 cannot act on intact Holliday junctions. The observed reaction of AtRECQ3 on the replication fork is in line with unwinding the lagging strand. On nicked Holliday junctions, which have not been intensively studied with RecQ helicases before, AtRECQ3, but not AtRECQ2, shows a clear preference for one unwinding mechanism. In addition, AtRECQ3 is much more efficient at catalyzing DNA strand annealing. Thus, AtRECQ2 and AtRECQ3 are likely to perform different tasks in the cell, and AtRECQ3 differs in its biochemical properties from all other eukaryotic RECQ helicases characterized so far.
Asunto(s)
Arabidopsis/enzimología , ADN Cruciforme/metabolismo , ADN de Plantas/metabolismo , RecQ Helicasas/metabolismo , Arabidopsis/genética , Replicación del ADN , Desoxirribonucleótidos/metabolismo , Datos de Secuencia Molecular , RecQ Helicasas/genética , RecQ Helicasas/aislamiento & purificaciónRESUMEN
The MUS81 endonuclease complex has been shown to play an important role in the repair of stalled or blocked replication forks and in the processing of meiotic recombination intermediates from yeast to humans. This endonuclease is composed of two subunits, MUS81 and EME1. Surprisingly, unlike other organisms, Arabidopsis (Arabidopsis thaliana) has two EME1 homologs encoded in its genome. AtEME1A and AtEME1B show 63% identity on the protein level. We were able to demonstrate that, after expression in Escherichia coli, each EME1 protein can assemble with the unique AtMUS81 to form a functional endonuclease. Both complexes, AtMUS81-AtEME1A and AtMUS81-AtEME1B, are not only able to cleave 3'-flap structures and nicked Holliday junctions (HJs) but also, with reduced efficiency, intact HJs. While the complexes have the same cleavage patterns with both nicked DNA substrates, slight differences in the processing of intact HJs can be detected. Our results are in line with an involvement of both MUS81-EME1 endonuclease complexes in DNA recombination and repair processes in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , ADN Cruciforme/metabolismo , Endonucleasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Secuencia de Bases , Cationes Bivalentes/farmacología , ADN Cruciforme/genética , Endonucleasas/aislamiento & purificación , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato/efectos de los fármacosRESUMEN
RecQ helicases play an important role in the maintenance of genomic stability in pro- and eukaryotes. This is highlighted by the human genetic diseases Werner, Bloom's and Rothmund-Thomson syndrome, caused by respective mutations in three of the five human RECQ genes. The highest numbers of RECQ homologous genes are found in plants, e.g. seven in Arabidopsis thaliana. However, only limited information is available on the functions of plant RecQ helicases, and no biochemical characterization has been performed. Here, we demonstrate that AtRECQ2 is a (d)NTP-dependent 3'-->5' DNA helicase. We further characterized its basal properties and its action on various partial DNA duplexes. Importantly, we demonstrate that AtRECQ2 is able to disrupt recombinogenic structures: by disrupting various D-loop structures, AtRECQ2 may prevent non-productive recombination events on the one hand, and may channel repair processes into non-recombinogenic pathways on the other hand, thus facilitating genomic stability. We show that a synthetic partially mobile Holliday junction is processed towards splayed-arm products, possibly indicating a branch migration function for AtRECQ2. The biochemical properties defined in this work support the hypothesis that AtRECQ2 might be functionally orthologous to the helicase part of the human RecQ homologue HsWRN.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , ADN de Plantas/química , RecQ Helicasas/fisiología , Recombinación Genética/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , ADN Cruciforme/química , ADN Cruciforme/metabolismo , ADN de Plantas/metabolismo , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , RecQ Helicasas/química , RecQ Helicasas/aislamiento & purificaciónRESUMEN
We present biochemical evidence for the occurrence of a 250-kD multifunctional acetyl-coenzyme A carboxylase in barley (Hordeum vulgare) mitochondria. Organelles from 6-d-old barley seedlings were purified by differential centrifugation and Percoll density gradient centrifugation. Upon analysis by two-dimensional Blue-native (BN)/SDS-PAGE, an abundant 250-kD protein can be visualized, which runs at 500 kD on the native gel dimension. A similar 500-kD complex is present in etioplasts from barley. The mitochondrial 250-kD protein is biotinylated as indicated by specific reaction with an antibody directed against biotin. Peptide sequence analysis by electrospray ionization tandem mass spectrometry of the 250-kD proteins from both organellar fractions revealed amino acid sequences that are 100% identical to plastidic acetyl-coenzyme A carboxylase from wheat (Triticum aestivum). The 500-kD complex was also detected in wheat mitochondria, but is absent in mitochondrial fractions from Arabidopsis. Specific acetyl-coenzyme A carboxylation activity in barley mitochondria is higher than in etioplasts, suggesting an important role of mitochondria in fatty acid biosynthesis. Functional implications are discussed.