RESUMEN
While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity.
Asunto(s)
Proteínas Bacterianas/química , Deinococcus/enzimología , Desoxirribonucleasa (Dímero de Pirimidina)/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/fisiología , Clonación Molecular , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de Proteína , Relación Estructura-ActividadRESUMEN
DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-situ-repaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Reparación del ADN/fisiología , ADN de Plantas/química , ADN de Cadena Simple/química , Desoxirribodipirimidina Fotoliasa/química , Dímeros de Pirimidina/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Criptocromos , Cristalografía por Rayos X , Daño del ADN/fisiología , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , ADN de Plantas/genética , ADN de Plantas/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Dímeros de Pirimidina/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
Cryptochromes are almost ubiquitous blue-light receptors and act in several species as central components of the circadian clock. Despite being evolutionary and structurally related with DNA photolyases, a class of light-driven DNA-repair enzymes, and having similar cofactor compositions, cryptochromes lack DNA-repair activity. Cryptochrome 3 from the plant Arabidopsis thaliana belongs to the DASH-type subfamily. Its crystal structure determined at 1.9 Angstroms resolution shows cryptochrome 3 in a dimeric state with the antenna cofactor 5,10-methenyltetrahydrofolate (MTHF) bound in a distance of 15.2 Angstroms to the U-shaped FAD chromophore. Spectroscopic studies on a mutant where a residue crucial for MTHF-binding, E149, was replaced by site-directed mutagenesis demonstrate that MTHF acts in cryptochrome 3 as a functional antenna for the photoreduction of FAD.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Ácido Fólico/análogos & derivados , Complejos de Proteína Captadores de Luz/metabolismo , Alanina , Secuencia de Aminoácidos , Sitios de Unión , Criptocromos , Electrones , Flavina-Adenina Dinucleótido/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Glutamina , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estructura Secundaria de Proteína , Espectrofotometría , Electricidad Estática , Relación Estructura-ActividadRESUMEN
The growth of branched actin networks powers cell-edge protrusions and motility. A heterogeneous density of actin, which yields to a tunable cellular response, characterizes these dynamic structures. We study how actin organization controls both the rate and the steering during lamellipodium growth. We use a high-resolution surface structuration assay combined with mathematical modeling to describe the growth of a reconstituted lamellipodium. We demonstrate that local monomer depletion at the site of assembly negatively impacts the network growth rate. At the same time, network architecture tunes the protrusion efficiency, and regulates the rate of growth. One consequence of this interdependence between monomer depletion and network architecture effects is the ability of heterogeneous network to impose steering during motility. Therefore, we have established that the general principle, by which the cell can modulate the rate and the direction of a protrusion, is by varying both density and architecture of its actin network.Protrusive cellular structures contain a heterogeneous density of actin, but whether this influences motility is not known. Using an in vitro system and modelling, here the authors show that local actin monomer depletion and network architecture can tune the rate of network growth to impose steering during motility.
Asunto(s)
Citoesqueleto de Actina/fisiología , Actinas/fisiología , Movimiento Celular/fisiología , Seudópodos/fisiología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Microscopía Fluorescente , Modelos Biológicos , Músculo Esquelético/química , Polimerizacion , ConejosRESUMEN
Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome-antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 A. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
Asunto(s)
Arabidopsis/metabolismo , Flavoproteínas/química , Proteínas de Arabidopsis , Catálisis , Clonación Molecular , Criptocromos , Cristalización , Cristalografía por Rayos X , Desoxirribodipirimidina Fotoliasa/química , Dimerización , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Flavina-Adenina Dinucleótido/química , Luz , Metilenotetrahidrofolato Deshidrogenasa (NAD+)/química , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes , Espectrofotometría , Relación Estructura-Actividad , Sincrotrones , Rayos Ultravioleta , Difracción de Rayos XRESUMEN
Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31â Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09â Å, ß = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47â Å, ß = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.
Asunto(s)
Deinococcus/enzimología , Endonucleasas/metabolismo , Cristalización , Cristalografía por Rayos X , Endonucleasas/químicaRESUMEN
X-ray crystallographic and functional analysis of the class I DNA photolyase from Thermus thermophilus revealed the binding of flavin mononucleotide (FMN) as an antenna chromophore. The binding mode of FMN closely coincides with the binding of a deazaflavin-like chromophore in the related class I DNA photolyase from Anacystis nidulans. Compared to the R46E mutant, which lacks a conserved arginine in the binding site for the antenna chromophore, the FMN-comprising holophotolyase exhibits an eightfold higher activity at 450 nm. The facile incorporation of the flavin cofactors 8-hydroxy-deazariboflavin and 8-iodo-8-demethyl-riboflavin into the binding site for the antenna chromophore paves the way for wavelength-tuning of the activity spectra of DNA photolyases by using synthetic flavins.
Asunto(s)
Productos Biológicos/química , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Thermus thermophilus/enzimología , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Desoxirribodipirimidina Fotoliasa/aislamiento & purificación , Flavinas/química , Flavinas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
DNA photolyases use light energy to repair DNA that comprises ultraviolet-induced lesions such as the cis-syn cyclobutane pyrimidine dimers (CPDs). Here we report the crystal structure of a DNA photolyase bound to duplex DNA that is bent by 50 degrees and comprises a synthetic CPD lesion. This CPD lesion is flipped into the active site and split there into two thymines by synchrotron radiation at 100 K. Although photolyases catalyze blue light-driven CPD cleavage only above 200 K, this structure apparently mimics a structural substate during light-driven DNA repair in which back-flipping of the thymines into duplex DNA has not yet taken place.