13 research outputs found

    Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm

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    <p>Abstract</p> <p>Background</p> <p><it>Pyrococcus furiosus </it>Hjm (<it>Pfu</it>Hjm) is a structure-specific DNA helicase that was originally identified by <it>in vitro </it>screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Ī˜ (PolĪ˜), HEL308, and <it>Drosophila </it>Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that <it>Pfu</it>Hjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by <it>Pfu</it>Hjm will require its three-dimensional structure at atomic resolution.</p> <p>Results</p> <p>We determined the crystal structures of <it>Pfu</it>Hjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0ā€“2.7 ƅ. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of <it>Archaeoglobus fulgidus </it>Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolĪ˜, indicating the strong conformational conservation between archaea and eukarya.</p> <p>Conclusion</p> <p>The detailed comparison between our DNA-free <it>Pfu</it>Hjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.</p

    Comparative genomics of the tardigrades <i>Hypsibius dujardini</i> and <i>Ramazzottius varieornatus</i>

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    Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda

    Horizontal gene transfer in <i>H</i>. <i>dujardini</i>.

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    <p>(A) Horizontal gene transfer ratios in various metazoa. For a set of assembled arthropod and nematode genomes, genes were repredicted ab initio with Augustus. Putative horizontal gene transfer (HGT) loci were identified using the HGT index for the longest transcript for each gene from the new and the ENSEMBL reference gene sets. In most species, the ab initio gene sets had elevated numbers of potential HGT loci compared to their ENSEMBL representations. Data available at <a href="https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig2A_HGT-content-in-metazoa.txt" target="_blank">https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig2A_HGT-content-in-metazoa.txt</a>. (B) Classification of HGT candidates in <i>H</i>. <i>dujardini</i>. Classification of the initial HGT candidates identified in <i>H</i>. <i>dujardini</i> by their phylogenetic annotation (prokaryotic, nonmetazoan eukaryotic, viral, complex HGT, and likely non-HGT metazoan and complex), their support in RNA-Seq expression data, and the presence of a homologue in <i>R</i>. <i>varieornatus</i>. Data available at <a href="https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig2B_HGT_content_in_Hdujardini.csv" target="_blank">https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig2B_HGT_content_in_Hdujardini.csv</a>.</p

    Phylogeny of Ecdysozoa.

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    <p>(A) Phylogeny of 28 species from 5 phyla, based on 322 loci derived from whole genome sequences, and rooted with the lophotrochozoan outgroup. The labels on the nodes are Bayes proportions from PhyloBayes analysis / bootstrap proportions from Randomized Axelerated Maximum Likelihood (RAxML) maximum likelihood bootstraps / proportion of trees of individual loci supporting each bipartition. Note that different numbers of trees were assessed at each node, depending on the representation of the taxa at each locus. * indicates maximal support (Bayes proportion of 1.0 or RAxML bootstrap of 1.0). (B) Phylogeny of 36 species from 8 phyla, based on 71 loci derived using PhyloBayes from whole genome and transcriptome sequences, and rooted with the lophotrochozoan outgroup. All nodes had maximal support in Bayes proportions and RAxML bootstrap, except those labeled (Bayes proportion, * = 1.0 / RAxML bootstrap).</p

    The genomes of <i>H</i>. <i>dujardini</i> and <i>R</i>. <i>varieornatus</i>.

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    <p>(A) Linkage conservation but limited synteny between <i>H</i>. <i>dujardini</i> and <i>R</i>. <i>varieornatus</i>. Whole genome alignment was performed with Murasaki [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002266#pbio.2002266.ref061" target="_blank">61</a>]. The left panel shows the whole genome alignment. Similar regions are linked by a line colored following a spectrum based on the start position in <i>R</i>. <i>varieornatus</i>. To the right is a realignment of the initial segment of <i>H</i>. <i>dujardini</i> scaffold0001 (lower), showing matches to several portions of <i>R</i>. <i>varieornatus</i> Scaffold0002 (above), illustrating the several inversions that must have taken place. The histograms show pairwise nucleotide sequence identity between these 2 segments. (B) Increased intron span in <i>H</i>. <i>dujardini</i>. <i>H</i>. <i>dujardini</i> genes are longer because of expanded introns. Frequency histogram of log<sub>2</sub> ratio of intron span per gene in 4,728 <i>H</i>. <i>dujardini</i> genes compared to their orthologues in <i>R</i>. <i>varieornatus</i>. Outliers are defined as genes in <i>H</i>. <i>dujardini</i> whose coding sequences (CDSs) are 20% longer (long outliers; orange; 576 genes) or 20% shorter (short outliers; black; 294 genes) than their orthologues in <i>R</i>. <i>varieornatus</i>. Data available at <a href="https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig1B_HDUJA_RVARI.gene_structure_matrix.ONE-TO-ONE.txt" target="_blank">https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig1B_HDUJA_RVARI.gene_structure_matrix.ONE-TO-ONE.txt</a>. (C) Gene neighborhood conservation between <i>H</i>. <i>dujardini</i> and <i>R</i>. <i>varieornatus</i>. To test conservation of gene neighborhoods, we asked whether genes found together in <i>H</i>. <i>dujardini</i> were also found close together in <i>R</i>. <i>varieornatus</i>. Taking the set of genes on each long <i>H</i>. <i>dujardini</i> scaffold, we identified the locations of the reciprocal best Basic Local Alignment Search Tool (BLAST) hit orthologues in <i>R</i>. <i>varieornatus</i> and counted the maximal proportion mapping to 1 <i>R</i>. <i>varieornatus</i> scaffold. <i>H</i>. <i>dujardini</i> scaffolds were binned and counted by this proportion. As short scaffolds, with fewer genes, might bias this analysis, we performed analyses independently on scaffolds with >10 genes and scaffolds with >20 genes. Data available at <a href="https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig1C_Gene-neighborhoods-conservation.txt" target="_blank">https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig1C_Gene-neighborhoods-conservation.txt</a>.</p

    The genomics of anhydrobiosis in tardigrades.

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    <p>(A) Gene losses in Hypsibiidae. Gene losses were detected by mapping to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using KEGG Automatic Annotation Server (KAAS) and validated by Basic Local Alignment Search Tool (BLAST) TBLASTN search of KEGG orthologue gene amino acid sequences. Light blue and gray boxes indicate genes conserved and lost in both tardigrades, respectively. Furthermore, purple boxes represent genes retained in only 1 species, and red boxes represent genes that have been detected as horizontal gene transfer (HGT). The numbers on the top right of the boxes indicate copy numbers of multiple copy genes in <i>H</i>. <i>dujardini</i>. Genes annotated as CASP3 and CDC25A have contradicting annotation with KAAS and Swiss-Prot; however, the KAAS annotation was used. (B) Differential gene expression in tardigrades on entry to the anhydrobiotic state. The transcript per million (TPM) expression for each sample was calculated using Kallisto, and the fold change between active and tun and the TPM expression in the tun state were plotted. Genes that likely contribute to anhydrobiosis were colored. Genes that had an orthologue in the other species are plotted as circles; other genes are plotted as triangles. Data available at <a href="https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig3B_gene-expression-DEGs-FCandTUN-annotated.txt" target="_blank">https://github.com/abs-yy/Hypsibius_dujardini_manuscript/blob/master/data/Fig3B_gene-expression-DEGs-FCandTUN-annotated.txt</a>. AMPK, 5' adenosine monophosphate-activated protein kinase; ATM, ataxia-telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; BER, base excision repair; BSC1, bypass of stop codon protein 1; CAHS, cytosolic abundant heat soluble; CASP8, caspase 8; CATE, catalase; CBF, C-repeat-binding factor; CHK1/2, checkpoint kinase 1/2; Dsup, damage suppressor; GST, glutathione S-transferase; HR, homologous recombination; HSP, heat shock protein; MAHS, mitochondrial abundant heat soluble protein; MMR, mismatch repair; mTOR, mechanistic target of rapamycin; NER, nucleotide excision repair; NHEJ, nonhomologous end joining; RvLEAM, late embryogenesis abundant protein mitochondrial; PHD, plant homeodomain; SAHS, secretory abundant heat soluble; SIRT1, sirtuin 1; SOD, superoxide dismutase; TSC1/2, tuberous sclerosis 1/2; VHL, von Hippel-Lindau tumor suppressor.</p

    The position of tardigrada in Ecdysozoa.

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    <p>(A) HOX genes in tardigrades and other Ecdysozoa. HOX gene losses in Tardigrada and Nematoda. HOX gene catalogues of tardigrades and other Ecdysozoa were collated by screening ENSEMBL Genomes and WormBase Parasite. HOX orthology groups are indicated by different colors. Some ā€œmissingā€ HOX loci were identified by Basic Local Alignment Search Tool (BLAST) search of target genomes (indicated by vertical striping of the affected HOX). ā€œ?ā€ indicates that presence/absence could not be confirmed because the species was surveyed by PCR or transcriptomics; loci identified by PCR or transcriptomics are indicated by a dotted outline. ā€œXā€ indicates that orthologous HOX loci were not present in the genome of that species. Some species have duplications of loci mapping to 1 HOX group, and these are indicated by boxes with dashed outlines. The relationships of the species are indicated by the cladogram to the left, and circles on this cladogram indicate Dollo parsimony mapping of events of HOX group loss on this cladogram. Circles are colored congruently with the HOX loci. (B) Evolution of gene families under different hypotheses of tardigrade relationships. Tardigrades share more gene families with Arthropoda than with Nematoda. In this network, derived from the OrthoFinder clustering at inflation value 1.5, nodes represent species (0: <i>Anopheles gambiae</i>, 1: <i>Apis mellifera</i>, 2: <i>Acyrthosiphon pisum</i>, 3: <i>Ascaris suum</i>, 4: <i>Brugia malayi</i>, 5: <i>Bursaphelenchus xylophilus</i>, 6: <i>Caenorhabditis elegans</i>, 7: <i>Cimex lectularius</i>, 8: <i>Capitella teleta</i>, 9: <i>Dendroctonus ponderosae</i>, 10: <i>Daphnia pulex</i>, 11: <i>Hypsibius dujardini</i>, 12: <i>Ixodes scapularis</i>, 13: <i>Meloidogyne hapla</i>, 14: <i>Nasonia vitripennis</i>, 15: <i>Octopus bimaculoides</i>, 16: <i>Priapulus caudatus</i>, 17: <i>Pediculus humanus</i>, 18: <i>Plectus murrayi</i>, 19: <i>Pristionchus pacificus</i>, 20: <i>Plutella xylostella</i>, 37: <i>Ramazzottius varieornatus</i>, 22: <i>Solenopsis invicta</i>, 23: <i>Strigamia maritima</i>, 24: <i>Tribolium castaneum</i>, 25: <i>Trichuris muris</i>, 26: <i>Trichinella spiralis</i>, 27: <i>Tetranychus urticae</i>, 38: <i>Drosophila melanogaster</i>). The thickness of the edge connecting 2 nodes is weighted by the count of shared occurrences of both nodes in OrthoFinder-clusters. Links involving <i>H</i>. <i>dujardini</i> (red) and <i>R</i>. <i>varieornatus</i> (orange) are colored. The inset box on the lower right shows the average weight of edges between each phylum and both Tardigrades, normalized by the maximum weight (i.e., count of co-occurrences of Tardigrades and the annelid <i>C</i>. <i>teleta</i>)" (C) Gene family birth synapomorphies at key nodes in Ecdysozoa under 2 hypotheses: Tardigrada+Nematoda versus Tardigrada+Arthropoda. Each graph shows the number of gene families at the specified node inferred using Dollo parsimony from OrthoFinder clustering at inflation value 1.5. Gene families are grouped by the proportion of taxa above that node that contain a member. Note that to be included as a synapomorphy of a node, a gene family must contain proteins of at least 1 species of each child node of the node in question, and thus, there are no synapomorphies with <0.3 proportional proteome coverage in Nematoda and <0.2 in Arthropoda, and all synapomorphies of Tardigrada have 1.0 representation. (D) Gene family birth synapomorphies for Tardigrada+Arthropoda (grey) and Tardigrada+Nematoda (yellow) for OrthoFinder clusterings performed at different Markov Cluster Algorithm (MCL) inflation parameters.</p
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