Transposon Excision from an Atypical Site: A Mechanism of Evolution of Novel Transposable Elements

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3′ to 5′ trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations

Direct observation of DNA threading in flap endonuclease complexes

Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted Vshaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “flycasting, thread, bend and barb” mechanis

The Contribution of Coevolving Residues to the Stability of KDO8P Synthase

The evolutionary tree of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes a key step in the biosynthesis of bacterial endotoxin, is evenly divided between metal and non-metal forms, both having similar structures, but diverging in various degrees in amino acid sequence. Mutagenesis, crystallographic and computational studies have established that only a few residues determine whether or not KDO8PS requires a metal for function. The remaining divergence in the amino acid sequence of KDO8PSs is apparently unrelated to the underlying catalytic mechanism.The multiple alignment of all known KDO8PS sequences reveals that several residue pairs coevolved, an indication of their possible linkage to a structural constraint. In this study we investigated by computational means the contribution of coevolving residues to the stability of KDO8PS. We found that about 1/4 of all strongly coevolving pairs probably originated from cycles of mutation (decreasing stability) and suppression (restoring it), while the remaining pairs are best explained by a succession of neutral or nearly neutral covarions.Both sequence conservation and coevolution are involved in the preservation of the core structure of KDO8PS, but the contribution of coevolving residues is, in proportion, smaller. This is because small stability gains or losses associated with selection of certain residues in some regions of the stability landscape of KDO8PS are easily offset by a large number of possible changes in other regions. While this effect increases the tolerance of KDO8PS to deleterious mutations, it also decreases the probability that specific pairs of residues could have a strong contribution to the thermodynamic stability of the protein

Mu Insertions Are Repaired by the Double-Strand Break Repair Pathway of Escherichia coli

Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps

Trace analysis of environmental matrices by large-volume injection and liquid chromatography-mass spectrometry

The time-honored convention of concentrating aqueous samples by solid-phase extraction (SPE) is being challenged by the increasingly widespread use of large-volume injection (LVI) liquid chromatography–mass spectrometry (LC–MS) for the determination of traces of polar organic contaminants in environmental samples. Although different LVI approaches have been proposed over the last 40 years, the simplest and most popular way of performing LVI is known as single-column LVI (SC-LVI), in which a large-volume of an aqueous sample is directly injected into an analytical column. For the purposes of this critical review, LVI is defined as an injected sample volume that is ≥10% of the void volume of the analytical column. Compared with other techniques, SC-LVI is easier to set up, because it requires only small hardware modifications to existing autosamplers and, thus, it will be the main focus of this review. Although not new, SC-LVI is gaining acceptance and the approach is emerging as a technique that will render SPE nearly obsolete for many environmental applications.In this review, we discuss: the history and development of various forms of LVI; the critical factors that must be considered when creating and optimizing SC-LVI methods; and typical applications that demonstrate the range of environmental matrices to which LVI is applicable, for example drinking water, groundwater, and surface water including seawater and wastewater. Furthermore, we indicate direction and areas that must be addressed to fully delineate the limits of SC-LVI

The wonders of flap endonucleases: structure, function, mechanism and regulation.

Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication

Africa and the global carbon cycle

The African continent has a large and growing role in the global carbon cycle, with potentially important climate change implications. However, the sparse observation network in and around the African continent means that Africa is one of the weakest links in our understanding of the global carbon cycle. Here, we combine data from regional and global inventories as well as forward and inverse model analyses to appraise what is known about Africa's continental-scale carbon dynamics. With low fossil emissions and productivity that largely compensates respiration, land conversion is Africa's primary net carbon release, much of it through burning of forests. Savanna fire emissions, though large, represent a short-term source that is offset by ensuing regrowth. While current data suggest a near zero decadal-scale carbon balance, interannual climate fluctuations (especially drought) induce sizeable variability in net ecosystem productivity and savanna fire emissions such that Africa is a major source of interannual variability in global atmospheric CO(2). Considering the continent's sizeable carbon stocks, their seemingly high vulnerability to anticipated climate and land use change, as well as growing populations and industrialization, Africa's carbon emissions and their interannual variability are likely to undergo substantial increases through the 21st century

Computing Highly Correlated Positions Using Mutual Information and Graph Theory for G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families

The role of inflammation in epilepsy.

Epilepsy is the third most common chronic brain disorder, and is characterized by an enduring predisposition to generate seizures. Despite progress in pharmacological and surgical treatments of epilepsy, relatively little is known about the processes leading to the generation of individual seizures, and about the mechanisms whereby a healthy brain is rendered epileptic. These gaps in our knowledge hamper the development of better preventive treatments and cures for the approximately 30% of epilepsy cases that prove resistant to current therapies. Here, we focus on the rapidly growing body of evidence that supports the involvement of inflammatory mediators-released by brain cells and peripheral immune cells-in both the origin of individual seizures and the epileptogenic process. We first describe aspects of brain inflammation and immunity, before exploring the evidence from clinical and experimental studies for a relationship between inflammation and epilepsy. Subsequently, we discuss how seizures cause inflammation, and whether such inflammation, in turn, influences the occurrence and severity of seizures, and seizure-related neuronal death. Further insight into the complex role of inflammation in the generation and exacerbation of epilepsy should yield new molecular targets for the design of antiepileptic drugs, which might not only inhibit the symptoms of this disorder, but also prevent or abrogate disease pathogenesis

Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of 5′ and 3′ Patches

BACKGROUND: Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3'-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. METHODOLOGY/PRINCIPLE FINDINGS: This paper re-examines heteroduplex-patch-strand polarity using phage lambda and the lambdadv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for lambda recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5' or 3' DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. CONCLUSIONS/SIGNIFICANCE: These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism