61 research outputs found

    Mobile genetic elements : in silico, in vitro, in vivo

    Get PDF
    Author Posting. Ā© The Author(s), 2015. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Molecular Ecology 25 (2016): 1027-1031, doi:10.1111/mec.13543.Mobile genetic elements (MGEs), also called transposable elements (TEs), represent universal components of most genomes and are intimately involved in nearly all aspects of genome organization, function, and evolution. However, there is currently a gap between fast-paced TE discovery in silico, stimulated by exponential growth of comparative genomic studies, and a limited number of experimental models amenable to more traditional in vitro and in vivo studies of structural, mechanistic, and regulatory properties of diverse MGEs. Experimental and computational scientists came together to bridge this gap at a recent conference, ā€œMobile Genetic Elements: in silico, in vitro, in vivo,ā€ held at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA.Research in the authorsā€™ laboratories is supported by NSF MCB-1121334 and NIH R01 GM111917 (I.A.), and by NIH R01 GM101989 and R21 AI117593 (P.R.)2016-11-3

    Deciphering the Roles of Multicomponent Recognition Signals by the AAA+ Unfoldase ClpX

    Get PDF
    ATP-dependent protein remodeling and unfolding enzymes are key participants in protein metabolism in all cells. How these often-destructive enzymes specifically recognize target protein complexes is poorly understood. Here, we use the well-studied AAA + unfoldase-substrate pair, Escherichia coli ClpX and MuA transposase, to address how these powerful enzymes recognize target protein complexes. We demonstrate that the final transposition product, which is a DNA-bound tetramer of MuA, is preferentially recognized over the monomeric apo-protein through its multivalent display of ClpX recognition tags. The important peptide tags include one at the C-terminus (ā€œC-tagā€) that binds the ClpX pore and a second one (enhancement or ā€œE-tagā€) that binds the ClpX N-terminal domain. We construct a chimeric protein to interrogate subunit-specific contributions of these tags. Efficient remodeling of MuA tetramers requires ClpX to contact a minimum of three tags (one C-tag and two or more E-tags), and that these tags are contributed by different subunits within the tetramer. The individual recognition peptides bind ClpX weakly (K[subscript D] > 70 Ī¼M) but impart a high-affinity interaction (K[subscript D] ~ 1.0 Ī¼M) when combined in the MuA tetramer. When the weak C-tag signal is replaced with a stronger recognition tag, the E-tags become unnecessary and ClpX's preference for the complex over MuA monomers is eliminated. Additionally, because the spatial orientation of the tags is predicted to change during the final step of transposition, this recognition strategy suggests how AAA + unfoldases specifically distinguish the completed ā€œend-stageā€ form of a particular complex for the ideal biological outcome.National Institutes of Health (U.S.) (Grants GM-49224 and AI-16892)National Institutes of Health (U.S.) (NIH Pre-Doctoral Training Grant T32GM007287

    The protein-protein interactions required for assembly of the Tn3 resolution synapse

    Get PDF
    The siteā€specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimerā€dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis (ā€œBartā€). Tn3 and Bart resolvases recognize different DNA motifs, via diverged Cā€terminal domains (CTDs). They also differ substantially at Nā€terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTDā€CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific ā€œR ā€ interface involving resolvase NTDs at all three dimerā€binding sites in res . Synapses containing mixtures of wildā€type Tn3 and Bart resolvase NTD dimers are recombinationā€defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa . We conclude that the Tn3 /Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimerā€dimer interaction required for catalysis

    Snapshots of a molecular swivel in action

    Get PDF
    Members of the serine family of site-specific recombinases exchange DNA strands via 180Ā° rotation about a central protein-protein interface. Modeling of this process has been hampered by the lack of structures in more than one rotational state for any individual serine recombinase. Here we report crystal structures of the catalytic domains of four constitutively active mutants of the serine recombinase Sin, providing snapshots of rotational states not previously visualized for Sin, including two seen in the same crystal. Normal mode analysis predicted that each tetramer's lowest frequency mode (i.e. most accessible large-scale motion) mimics rotation: two protomers rotate as a pair with respect to the other two. Our analyses also suggest that rotation is not a rigid body movement around a single symmetry axis but instead uses multiple pivot points and entails internal motions within each subunit

    Variable orthogonality of serine integrase interactions within the Ļ•C31 family

    Get PDF
    Serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases (Ļ•C31, Ļ•BT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases ā€“ RDF pairs. Ļ•C31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, Ļ•C31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate Ļ•C31 integrase. Ļ•BT1 and TG1 RDFs were noticeably more effective than Ļ•C31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase ā€“ RDF pair. The binding surface on the RDF is much more conserved than the binding surface on the integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase ā€“ RDF pairs. Additionally, the ability of a particular integraseā€™s preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches

    Meeting Report for Mobile DNA 2010

    Get PDF
    An international conference on mobile DNA was held 24-28 April 2010 in Montreal, Canada. Sponsored by the American Society for Microbiology, the conference's goal was to bring together researchers from around the world who study transposition in diverse organisms using multiple experimental approaches. The meeting drew over 190 attendees and most contributed through poster presentations, invited talks and short talks selected from poster abstracts. The talks were organized into eight scientific sessions, which ranged in topic from the evolutionary dynamics of mobile genetic elements to transposition reaction mechanisms. Here we present highlights from the platform sessions with a focus on talks presented by the invited speakers

    Structural basis for topological regulation of Tn3 resolvase

    Get PDF
    Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and Ī³Ī“ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit proteinā€“DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase

    Inter-subunit interactions that coordinate Rad51's activities

    Get PDF
    Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis

    Effect of a Perioperative, Cardiac Output-Guided Hemodynamic Therapy Algorithm on Outcomes Following Major Gastrointestinal Surgery A Randomized Clinical Trial and Systematic Review

    Get PDF
    Importance: small trials suggest that postoperative outcomes may be improved by the use of cardiac output monitoring to guide administration of intravenous fluid and inotropic drugs as part of a hemodynamic therapy algorithm.Objective: to evaluate the clinical effectiveness of a perioperative, cardiac outputā€“guided hemodynamic therapy algorithm.Design, setting, and participants: OPTIMISE was a pragmatic, multicenter, randomized, observer-blinded trial of 734 high-risk patients aged 50 years or older undergoing major gastrointestinal surgery at 17 acute care hospitals in the United Kingdom. An updated systematic review and meta-analysis were also conducted including randomized trials published from 1966 to February 2014.Interventions: patients were randomly assigned to a cardiac outputā€“guided hemodynamic therapy algorithm for intravenous fluid and inotrope (dopexamine) infusion during and 6 hours following surgery (n=368) or to usual care (n=366).Main outcomes and measures: the primary outcome was a composite of predefined 30-day moderate or major complications and mortality. Secondary outcomes were morbidity on day 7; infection, critical careā€“free days, and all-cause mortality at 30 days; all-cause mortality at 180 days; and length of hospital stay.Results: baseline patient characteristics, clinical care, and volumes of intravenous fluid were similar between groups. Care was nonadherent to the allocated treatment for less than 10% of patients in each group. The primary outcome occurred in 36.6% of intervention and 43.4% of usual care participants (relative risk [RR], 0.84 [95% CI, 0.71-1.01]; absolute risk reduction, 6.8% [95% CI, ?0.3% to 13.9%]; P?=?.07). There was no significant difference between groups for any secondary outcomes. Five intervention patients (1.4%) experienced cardiovascular serious adverse events within 24 hours compared with none in the usual care group. Findings of the meta-analysis of 38 trials, including data from this study, suggest that the intervention is associated with fewer complications (intervention, 488/1548 [31.5%] vs control, 614/1476 [41.6%]; RR, 0.77 [95% CI, 0.71-0.83]) and a nonsignificant reduction in hospital, 28-day, or 30-day mortality (intervention, 159/3215 deaths [4.9%] vs control, 206/3160 deaths [6.5%]; RR, 0.82 [95% CI, 0.67-1.01]) and mortality at longest follow-up (intervention, 267/3215 deaths [8.3%] vs control, 327/3160 deaths [10.3%]; RR, 0.86 [95% CI, 0.74-1.00]).Conclusions and relevance: in a randomized trial of high-risk patients undergoing major gastrointestinal surgery, use of a cardiac outputā€“guided hemodynamic therapy algorithm compared with usual care did not reduce a composite outcome of complications and 30-day mortality. However, inclusion of these data in an updated meta-analysis indicates that the intervention was associated with a reduction in complication rate
    • ā€¦
    corecore