Structural and mechanistic insight into DNA unwinding by Deinococcus radiodurans UvrD.

DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3'-5' direction and shows ATP-dependent 3'-5', and surprisingly also, 5'-3' helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD's 3'-5' and 5'-3' helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5'-3' helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo

Dynamics of human mitochondrial complex I assembly: Implications for neurodegenerative diseases

none5Neurons are extremely energy demanding cells and highly dependent on the mitochondrial oxidative phosphorylation (OXPHOS) system. Mitochondria generate the energetic potential via the respiratory complexes I to IV, which constitute the electron transport chain (ETC), together with complex V. These redox reactions release energy in the form of ATP and also generate reactive oxygen species (ROS) that are involved in cell signaling but can eventually lead to oxidative stress. Complex I (CI or NADH:ubiquinone oxidoreductase) is the largest ETC enzyme, containing 44 subunits and the main contributor to ROS production. In recent years, the structure of the CI has become available and has provided new insights into CI assembly. A number of chaperones have been identified in the assembly and stability of the mature holo-CI, although they are not part of its final structure. Interestingly, CI dysfunction is the most common OXPHOS disorder in humans and defects in the CI assembly process are often observed. However, the dynamics of the events leading to CI biogenesis remain elusive, which precludes our understanding of how ETC malfunctioning affects neuronal integrity. Here, we review the current knowledge of the structural features of CI and its assembly factors and the potential role of CI misassembly in human disorders such as Complex I Deficiencies or Alzheimer's and Parkinson's diseases.openGiachin G.; Bouverot R.; Acajjaoui S.; Pantalone S.; Soler-Lopez M.Giachin, G.; Bouverot, R.; Acajjaoui, S.; Pantalone, S.; Soler-Lopez, M

Conformational changes associated with ATP hydrolysis and nucleotide release.

<p>A-C. Domain movements. The AMPPNP-bound form is colored in red, while the apo-form is colored in blue. A. Upon ATP hydrolysis and nucleotide release, domain 2B along with the dsDNA rotates by ~15° and domain 1A and 1B by 8° relative to domain 2A. B. Close up view of the rotation of domain 2B and duplex DNA. C. Domains 1A and 1B undergo a 15° twist relative to domain 2A around the ssDNA axis (orange). D. Conformational changes occurring at the ssDNA gateway (circled in green). The linker between domains 2B and 2A adopts a short helix (α25) and loop in the AMPPNP-bound form and interacts tightly with the 3′-end of the ssDNA via Ser546, while it consists of an unstructured loop (dashed line) in the apo-form. In the AMPPNP form, the ssDNA gateway is more closed: the distance between the carboxyl oxygen of Phe65 (motif Ia) and the hydroxyl group of Ser546 is 4.5 Å in the AMPPNP-bound form versus 9.9 Å in the apo-form. The represented DNA corresponds to the AMPPNP bound form.</p

Crystal structures of <i>dr</i>UvrD-DNA complexes.

<p>A ribbon illustration of the AMPPNP-bound <i>dr</i>UvrD^FL is shown in A, the AMPPNP-bound <i>dr</i>UvrD^∆C form I is shown in B, the mixed AMPPNP-bound (red) and apo- (blue) <i>dr</i>UvrD^∆C form II is shown in C. The DNA and AMPPNP are shown in sticks. D-E. Large-scale conformational changes. D. Overlay of chains A (red) of <i>dr</i>UvrD^FL, <i>dr</i>UvrD^∆C form I and apo-<i>dr</i>UvrD^∆C form II, illustrating the large spiral movement of chains B colored respectively yellow, grey and blue. The DNA is shown as an orange ribbon. E. As in (D) but viewed down the DNA axis, and for clarity <i>dr</i>UvrD^∆C form I has been removed. </p

DNA binding ability and helicase activity of drUvrD mutants.

<p>Comparison of DNA binding ability and helicase activity of wild type (WT) and <i>dr</i>UvrD mutants: β-hairpin deletion mutant (ΔHairpin), and mutants of the GIG motif from domain 2B involved in dsDNA binding (G424T, G426T and double mutant G424T/G426T). A. DNA binding affinities (K_d values) of WT and mutant <i>dr</i>UvrD for either 3'-tailed (blue) or 5'-tailed (red) dsDNA determined by fluorescence anisotropy measurements. B. Helicase activity of WT and mutant <i>dr</i>UvrD (250 nM) on 3'-tailed (blue) or 5'-tailed (red) dsDNA (20 nM). Initial reaction rates were determined from reaction time courses and were normalized with respect to the activity of WT <i>dr</i>UvrD. Standard deviations are shown as vertical bars.</p

DNA binding of <i>dr</i>UvrD.

<p>Illustrations of <i>dr</i>UvrD binding to dsDNA with a 3′-ssDNA tail in form I (A,D and G), form II with AMPPNP bound (B, E and H) and in the apo-form of form II (C, F and I). A-C. Schematic diagrams (top) illustrating the translocation of form I (A), form II with AMPPNP bound (B) and the apo-form of form II (C) of <i>dr</i>UvrD^∆C along the ssDNA. The ssDNA nucleotides are illustrated as black bars and are numbered as in the crystal structures. The grey oval shape representing <i>dr</i>UvrD covers the nucleotides bound in the ssDNA binding pocket. Surface representations of the ssDNA binding pockets of these three forms of <i>dr</i>UvrD^∆C bound to ssDNA (orange sticks) are shown below. The important residues are labeled and the bases are numbered as in the schematic diagrams. D-F. Binding of <i>dr</i>UvrD^∆C to dsDNA in form I (D), form II with AMPPNP bound (E) and in the apo-form of form II (F). The dsDNA is illustrated in sticks with the translocated strand in grey. Domains of <i>dr</i>UvrD are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077364#pone-0077364-g002" target="_blank">Figure 2A</a>. The helices belonging to the HLH motifs and the β-hairpin structure (orange) are shown and labeled according to the secondary structure succession (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077364#pone.0077364.s005" target="_blank">Figure S1</a>). The positively charged residues in contact with dsDNA are illustrated in sticks and the GIG motif is indicated. The number of base-pairs formed between the ss-dsDNA junction and the contact point with the <i>dr</i>UvrD GIG motif is shown to the left of each panel. This number differs significantly between the two crystal forms. G-I. Schematic representation of <i>dr</i>UvrD's DNA binding in the different crystal structures as indicated below the models. The four protein-DNA contact points that are critical for the wrench-and-inchworm unwinding mechanism are indicated with circled numbers in all panels: HLH motifs interact with dsDNA (1), the β-hairpin motif with the ss-dsDNA junction (2), motif III with the ssDNA (3) and the ssDNA gateway with the exiting ssDNA (4). G. In AMPPNP bound Form I, contact points 1, 3 and 4 are tight. H. In AMPPNP bound Form II, <i>dr</i>UvrD's GIG motif (1) has slided along the DNA duplex and pushes the DNA junction against the β-hairpin motif (2), which now stacks tightly against the first base-pair. I. In the apo molecule of Form II, the ssDNA gateway (4) has opened and ssDNA exited the helicase. Domains of <i>dr</i>UvrD are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077364#pone-0077364-g002" target="_blank">Figure 2A</a>.</p

ATPase and helicase activity of <i>dr</i>UvrD.

<p>A. DNA-stimulated ATPase kinetic parameters of <i>dr</i>UvrD^FL and <i>dr</i>UvrD^∆C. B. Structure of DNA oligonucleotides used for helicase assay of <i>dr</i>UvrD. The fluorescein label is represented as a star. C.-D. Helicase activity of <i>dr</i>UvrD^FL on DNA substrates shown in (B). C. Table summarising the initial rates of unwinding of duplexed DNA containing 15 or 7 nucleotide ssDNA extensions at either the 3′ or 5′ ends and of blunt duplexed DNA, as indicated, and in the absence and presence of <i>dr</i>SSB (250 nM). The rates are given in base-pairs per min per UvrD helicase unit (bp/min/UvrD). D. Time course of <i>dr</i>UvrD unwinding of duplexed DNA containing 15 nucleotide ssDNA extensions at either the 3′ (red) or 5′ (black)-ends and of blunt (blue) duplexed DNA in the absence (full line) and presence (dotted line) of <i>dr</i>SSB (250 nM). Standard deviations are shown as vertical bars.</p

ssDNA translocase activity of <i>dr</i>UvrD.

<p>Translocase activity of <i>dr</i>UvrD was assayed using the streptavidin-displacement assay. A. Structure of DNA oligonucleotides used for <i>dr</i>UvrD translocase assay measuring streptavidin displacement from biotinylated DNA substrates. The fluorescein label is represented as a star and the biotin label as a circle. B. Time course of <i>dr</i>UvrD (250 nM) catalyzed streptavidin displacement from the 3′- (blue) and 5′- (red) ssDNA extensions of DNA oligonucleotides shown in (A). The fraction of released dsDNA (no longer bound to streptavidin) was quantified and plotted as a function of time. C. Translocase activity of <i>dr</i>UvrD (250 nM) on 5' tailed dsDNA (20 nM) as a function of time in the absence (left) and the presence (right) of <i>dr</i>SSB (250 nM). The reaction products were analyzed on a 10 % polyacrylamide TBE gel. Bands correspond to the fluorescein labeled reaction products: streptavidin-bound dsDNA (upper bands, corresponding to several biotin labeled oligonucleotides bound to streptavidin), released dsDNA (middle band) and unwound ssDNA (lower band). D. The bands shown in (C), resulting from the time course of streptavidin displacement from 5′- tailed dsDNA, were quantified and the fraction of streptavidin-bound (black), released dsDNA (red) and unwound ssDNA (blue) were plotted as a function of time for reactions carried out in the absence (full lines) and presence (dotted lines) of <i>dr</i>SSB (250 nM). Standard deviations are shown as vertical bars.</p

Domain organization of <i>dr</i>UvrD and structure of the various DNA oligonucleotides used for crystallization.

<p>A. Schematic representation of the domain structures of <i>dr</i>UvrD^FL and <i>dr</i>UvrD^∆C. B. Structure of DNA oligonucleotides used for crystallization with <i>dr</i>UvrD^FL and <i>dr</i>UvrD^∆C. The circles represent UvrD bound to the DNA as observed in our crystal structures. </p

Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex

International audienceTo eliminate specific or aberrant transcripts, eukaryotes use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC, and its human counterpart PAXT, are key players in this mechanism but inner workings of these complexes are not understood in sufficient detail. Here, we present an NMR structure of an MTREC scaffold protein Red1 helix-turn-helix domain bound to the Iss10 N-terminus and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1 - Ars2 complex explaining mutually exclusive interactions of hARS2 with various ED/EGEI/L motif-possessing RNA regulators, including hZFC3H1 of PAXT, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture