Orsolya BarabasUniversity of Geneva | UNIGE · Department of Molecular Biology
Orsolya Barabas
PhD
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74
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Introduction
Additional affiliations
January 2021 - present
November 2009 - December 2021
November 2005 - November 2009
Education
September 2001 - January 2004
September 2001 - October 2005
September 1996 - June 2001
Publications
Publications (74)
CRISPR–Cas is a bacterial defence system that can attack invading DNA to protect host cells, or help to insert DNA safely into the genome. Structures of this latter type of CRISPR–Cas system have now been visualized. High-resolution structures of CRISPR-associated transposon system.
Transposons are mobile genetic elements that drive evolution and adaptation throughout the tree of life. In bacteria, they often transfer antibiotic resistance genes and contribute to the emergence of multidrug‐resistant pathogens.
In this talk, I will present our recent discoveries on a group of transposons that efficiently propagate antibiotic re...
Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and enviro...
The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino aci...
Mobile genetic elements (MGEs) sequester and mobilize antibiotic resistance genes across bacterial genomes. Efficient and reliable identification of such elements is necessary to follow resistance spreading. However, automated tools for MGE identification are missing. Tyrosine recombinase (YR) proteins drive MGE mobilization and could provide marke...
Phosphoprotein phosphatase‐1 (PP1) is a key player in the regulation of phospho‐serine (pSer) and phospho‐threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well‐established activity control by...
The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design...
The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we es...
Transposase proteins mediate the movement of ‘parasitic’ DNA segments in genomes. A series of structures of a transposase catches it in action, and highlights how these proteins evolved for use in immune systems. A sequence of structures of a transposase enzyme in action.
Transposases move discrete pieces of DNA between genomic locations and had a profound impact on evolution. They drove the emergence of important biological functions and are the most frequent proteins encoded in modern genomes. Yet, the molecular principles of their actions have remained largely unclear. Here we review recent structural studies of...
Background: Tyrosine recombinases perform site-specific genetic recombination in bacteria and archaea. They safeguard genome integrity by resolving chromosome multimers, as well as mobilize transposons, phages and integrons, driving dissemination of genetic traits and antibiotic resistance. Despite their abundance and genetic impact, tyrosine recom...
Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and nonself nucleic acids and maintain genome integrity and are essential for fertility in a variety of organisms. In Caenorhabditis elegans, most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. Whil...
Protein phosphatase‐1 (PP1) drives a large amount of phosphoSer/Thr protein dephosphorylations in eukaryotes to counteract multiple kinases in signalling pathways. The phosphatase requires divalent metal cations for catalytic activity and contains iron naturally. Iron has been suggested to have an influence on PP1 activity through Fe2+ and Fe3+ oxi...
Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and non-self nucleic acids, maintain genome integrity, and are essential for fertility in a variety of organisms. In C. elegans most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes...
Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairi...
Movie S1. Conformational Rearrangements Required for Tetramerization and Target DNA Binding, Related to Figure 7
Morphing of the dimeric Tn1549 Int82N-CI DNA complex into a model of the proposed tetrameric Tn1549 Int82N-CI-target DNA ternary complex (modeled based on the λInt–COC’ post-cleavage synaptic complex structure, PDB ID: 1z19). Color code...
Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transp...
Rapid spread of resistance to vancomycin has generated difficult to treat bacterial pathogens worldwide. Though vancomycin resistance is often conferred by the conjugative transposon Tn1549, it is yet unclear whether Tn1549 moves actively between bacteria. Here we demonstrate, through development of an in vivo assay system, that a mini-Tn1549 can t...
The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via...
Quantification of XerH binding to difH variants based on EMSA experiments.The original gels are shown in Figure 3—figure supplement 1.DOI:
http://dx.doi.org/10.7554/eLife.19706.011
Results of the in vivo recombination assays.Colony counts and recombination rates are tabulated and their statistical analysis by Student’s t-test is shown.DOI:
http://dx.doi.org/10.7554/eLife.19706.012
List of oligonucleotides used in this study.
DOI:
http://dx.doi.org/10.7554/eLife.19706.022
Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected,...
Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the cryst...
Supplementary Figures 1-5 and Supplementary References.
Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB’s applications, here we determine the cryst...
The thermophilic fungus Chaetomium thermophilum holds great promise for structural biology. To increase the efficiency of its biochemical and structural characterization
and to explore its thermophilic properties beyond those of individual proteins, we obtained transcriptomics and proteomics
data, and integrated them with computational annotation m...
In bacteria, small RNAs (sRNAs) silence or activate target genes through base pairing with the mRNA, thereby modulating its translation. A central player in this process is the RNA chaperone Hfq, which facilitates the annealing of sRNAs with their target mRNAs. Hfq has two RNA-binding surfaces that recognize A-rich and U-rich sequences, and is beli...
Significance
Transposons are jumping genes that constitute a sizeable fraction of eukaryotic genomes. They drive genome evolution and can cause genetic diseases and cancer. Although transposons were first discovered in plants and much of our knowledge about them stems from plants, the most abundant human transposon, L1, has barely been investigated...
Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like
signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues
regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, u...
Piwi-interacting RNAs (piRNAs) are a gonad-specific class of small RNAs that associate with the Piwi clade of Argonaute proteins and play a key role in transposon silencing in animals. Since biogenesis of piRNAs is independent of the double-stranded RNA-processing enzyme Dicer, an alternative nuclease that can process single-stranded RNA transcript...
Transposons are mobile genetic elements that have shaped evolution and thus constitute much of modern genomes. Their movements cause diseases, change regulatory patterns and spread antibiotic resistance. Single‐stranded deoxyribonucleic acid (ssDNA) transposition, is carried out by a unique transposase protein that recognises a hairpin structure fo...
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of fou...
Genes present in only certain strains of a bacterial species can strongly affect cellular phenotypes and evolutionary potentials. One segment that seemed particularly rich in strain-specific genes was found by comparing the first two sequenced Helicobacter pylori genomes (strains 26695 and J99) and was named a "plasticity zone".
We studied the natu...
Target site choice is a complex and poorly understood aspect of DNA transposition despite its importance in rational transposon-mediated gene delivery. Though most transposons choose target sites essentially randomly or with some slight sequence or structural preferences, insertion sequence IS608 from Helicobacter pylori, which transposes using sin...
Most dUTP pyrophosphatases (dUTPases) are homotrimers with interfaces formed between subunit surfaces, in the central channel, and by C-terminal beta-strand swapping. Analysis of intersubunit interactions reveals an important cohesive role for the C-terminus. This is reflected in the crystal structure of fruitfly dUTPase displaying a dimeric organi...
dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog,...
C1r is a modular serine protease which is the autoactivating component of the C1 complex of the classical pathway of the complement system. We have determined the first crystal structure of the entire active catalytic region of human C1r. This fragment contains the C-terminal serine protease (SP) domain and the preceding two complement control prot...
dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicr...
Bacterial insertion sequences (IS) play an important role in restructuring their host genomes. IS608, from Helicobacter pylori, belongs to a newly recognized and widespread IS group with a unique transposition mechanism. We have reconstituted the entire set of transposition cleavage and strand transfer reactions in vitro and find that, unlike any o...
The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent pr...
Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker w...
The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription,
and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained
obscured by the N- and C-terminal flexible segments of dUTPase and the linker region co...
Deoxyuridine 5'-triphosphate nucleotidohydrolase from Mason-Pfizer monkey retrovirus (M-PMV dUTPase) is a betaretroviral member of the dUTPase enzyme family. In the mature M-PMV virion, this enzyme is present as the C-terminal domain of the fusion protein nucleocapsid-dUTPase. The homotrimeric organization characteristic of dUTPases is retained in...
dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic info...
dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses...
Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody agains...
The aim of our study was to synthesize vinylic and pyrido-fused pyridazines with a spirano moiety and to investigate their stereochemistry by spectroscopic and HPLC analyses. The vinylic compounds 5 were obtained by Knoevenagel condensation of cyclohexylidene malonates 1 with pyridazinecarbaldehyde 2. Compound 5b exhibits geometric isomerism identi...
On heating with dialkyl-acetylenedicarboxylates in DMF condensed [1,2,4]-triazolo[4,3-b] pyridazine-6(5H)-one-3(2H)-thiones undergo unprecedented ring transformations yielding novel tetracyclic 1,3-diazepines and thiazolotriazole derivatives depending on the applied reaction temperature. The observed substrate selectivity was interpreted on the bas...
Cyclization of the title compounds with sodium ethoxide may proceed in various ways to afford differently fused pyridazino ring systems. Theoretical considerations based on frontier molecular orbital (FMO) analysis of 4-chloro-5-hydroxyalkylamino-6-nitropyridazinones were in agreement with experimental results in most cases. Formation of pyridazino...
Different ferrocenyl-substituted hydrazones of heteroaryl/arylhydrazines (1a–e, 3 and 10) were reacted with dimethyl fumarate and gave two epimeric pairs of pyrazolidines (t,c-2a and t,c-2c), pyrazolines (5a,c, 6, 11), pyrazoles (8, 12) and condensed triazoles (4a, 7). On treatment with dicyanodichloroquinone (DDQ) the pyrido[2,3-d]pyridazinyl-subs...
A systematic study of crystal packing in a series of structures is presented: an isostructural triplet of an optically active compound {(R)-2-(6,7-diethoxy-1,2,3,4-tetrahydro-1-isoquinolidene)-2-[2-hydroxy-3-(4-morpholinyl)propyl]mercaptoacetonitrile hydrochloride, C22H32ClN3O4S} 1, its racemate 2 and their achiral dehydroxy parent compound {2-(6,7...
Fehérjekrisztallográfia, mágneses magrezonancia-spektroszkópia és molekulamodellezés segítségével vizsgáltuk az összefüggéseket néhány metalloprotein, valamint egy új típusú, rendezetlen fehérje szerkezete és működése között. Hatékony módszert fejlesztettünk ki a reakcióút kvantummechanikai számítására enzimekben. Tisztáztuk a DNS javításában fonto...
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