Epigenetic deregulation of DNA repair and its potential for therapy.

Epigenetic silencing of essential components of DNA repair pathways is a common event in many tumor types, and comprise O6-methylguanine-DNA methyltransferase (MGMT), human mut L homolog 1 (hMLH1), Werner syndrome gene (WRN), breast cancer susceptibility gene 1 (BRCA1), and genes of the Fanconi anemia pathway. Most interestingly, some of these alterations become the Achilles heel of the affected tumors upon treatment with certain classes of anticancer agents. That is, patients whose tumors carry such defects can be stratified for respective therapy rendering some classic DNA damaging agents, such as alkylators or DNA crosslinking agents, into "targeted therapies." Here we review some of the affected repair pathways that, when inactivated, sensitize the tumors to specific drugs and are thus exploitable for individualized therapy.

Innate immunity: cytoplasmic DNA sensing by the AIM2 inflammasome.

Cytoplasmic double-stranded DNA triggers cell death and secretion of the pro-inflammatory cytokine IL-1beta in macrophages. Recent reports now describe the mechanism underlying this observation. Upon sensing of DNA, the HIN-200 family member AIM2 triggers the assembly of the inflammasome, culminating in caspase-1 activation, IL-1beta maturation and pyroptotic cell death.

A genetic isolation gradient of populations of the Balearic green toad (Bufo balearicus) follows rising eastward fragmentation of the rural landscapes on the island of Menorca.

We present the first approach to the genetic diversity and structure of the Balearic toad (Bufo balearicus Boettger, 1880) for the island of Menorca. Forty-one individ- uals from 21 localities were analyzed for ten microsatellite loci. We used geo-refer- enced individual multilocus genotypes and a model-based clustering method for the inference of the number of populations and of the spatial location of genetic dis- continuities between those populations.¦Only six of the microsatellites analyzed were polymorphic. We revealed a northwest- ern area inhabited by a single population with several well-connected localities and another set of populations in the southeast that includes a few unconnected small units with genetically significant differences among them as well as with the individ- uals from the northwest of the island. The observed fragmentation may be explained by shifts from agricultural to tourism practices that have been taking place on the island of Menorca since the 1960s. The abandonment of rural activities in favor of urbanization and concomitant service areas has mostly affected the southeast of the island and is currently threatening the overall geographic connectivity between the different farming areas of the island that are inhabited by the Balearic toad.

Functional analysis of human POT1 and RPA : insights into the role of single stranded DNA binding proteins at telomeres

Abstract Telomeres, the natural ends of chromosomes, need to be protected from chromosome end fusions, aberrant homologous recombination and degradation. In humans, chromosome ends are specified through arrays of tandemly repeated 5'-TTAGGG-3' hexamers, ending in a 3' overhang. A complex formed by the six proteins TRF1, TRF2, hRap1, TIN2, TPP1 and POT1 specifically assocìates with and protects telomeres. Telomeres are maintained by semiconservative DNA replication and by a specialized reverse transcriptase, telomerase, that carries an RNA subunit which templates new telomeric repeat synthesis. The telomeric single stranded (ss) DNA binding protein POT1 protects the telomeric 3' overhang and modulates telomerase-mediated telomere elongation. It is possible that POT1 also influences DNA synthesis during semiconservative DNA replication, which is initiated by the DNA polymerase alpha-primase complex. The heterotrimeric ss DNA-binding protein RPA plays essential roles during DNA replication. RPA binds to ss DNA with high affinity in order to stabilize ss DNA and facilitate nascent strand synthesis at the replication fork. Here we investigate how the two proteins RPA and POT1 contribute to telomere maintenance by regulating semi-conservative DNA replication and telomerase. Using chromatin immunoprecipitation experiments, we show that RPA associates with telomeres during S-phase. Analysis of telomere structure in cells shRNA-depleted for RPA and POT1 reveals that loss of RPA and POT1 causes exposure of single-stranded DNA at telomeres, suggestive of incomplete DNA replication. Biochemical experiments using purified recombinant POT1 and RPA show that saturating telomeric oligonucleotides with POT1 or RPA reduces the primase activity of the DNA polymerase alpha-primase complex and the overall activity of telomerase. POT1 and RPA also increase the primer extension by DNA polymerase alpha-primase complex and the processivity of telomerase under certain conditions, although POT1 increases the activities to a greater extent than RPA. We propose that POT1 is required for proper replication of the lagging strand of telomeres and that some phenotypes observed in POT1-depleted cells may stern from incomplete DNA replication rather than de-protection of the single-stranded overhang. Résumé Les télomères, les extrémités normales des chromosomes linéaires, doivent être protégés des fusions chromosomiques, d'événements de recombinaison homologue aberrants et de phénomènes de dégradation. Chez l'Homme, les extrémités des chromosomes sont constitués d'ADN double brin répétitif de séquence 5'-TTAGGG-3', d'une extension simple brin 3' sortante et d'un complexe protéique formé des six facteurs TRF1, TRF2, hRap1, TIN2, TPP1 et POT1 qui, s'associant à cette séquence, protègent l'ADN télomèrique. Les télomères sont maintenus par la télomérase, une transcriptase inverse capable d'allonger l'extension 3' sortante télomérique. POT1 lie l'ADN simple brin télomérique et module l'élongation des télomères par la télomérase. POT1 pourrait en théorie également influencer la réplication semi-conservative de l'ADN. L'ADN-polymérase Pal alpha-primase amorce et initie la synthèse d'ADN. Pendant la réplication, l'ADN simple brin est stabilisé par RPA, un complexe hétérotrimèrique qui lie l'ADN simple brin. RPA facilite la synthèse du brin naissant à la fourche de réplication. Ici nous avons étudié comment ces deux protéines qui lient l'ADN simple brin, RPA et POT1, régulent la réplication des télomères par la télomérase et la machinerie classique de réplication de l'ADN. Par immunoprécipitation de chromatine (ChIP), nous montrons que RPA est localisé aux télomères lors de la phase S du cycle cellulaire. De plus, l'analyse de la structure des télomeres indique que !a perte de RPA ou de POT1 conduit à l'apparition d'ADN simple brin télomérique, suggérant une réplication incomplète de l'ADN télomérique in vivo. Par une approche complémentaire biochimique utilisant les protéines POT1 et RPA recombinantes purifiées, nous montrons également que la liaison de POT1 ou de RPA à des oligonucléotides télomériques bloque l'activité primase du complexe polymérase alpha/primase et réduit l'activité télomérase sur ces substrats. En revanche, leur liaison augmente l'activité ADN-polymérase du complexe polymérase alpha/primase, ainsi que fa processivité de la télomérase dans certaines conditions, POT1 étant le plus efficace des deux facteurs. Nous proposons que POT1 est nécessaire à la réplication du brin retardé au niveau des télomères, ce qui suggère que certains phénotypes des cellules déplétés en POT1 puissent résulter d'une réplication incomplète de l'ADN télémétrique plutôt que d'une déprotection de l'extrémité sortante des télomères.

Identification of an agrin mutation that causes congenital myasthenia and affects synapse function.

We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases.

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.

Fragmentation energy in rock avalanches

La fragmentation est un des mécanismes opérant lors d'avalanche rocheuses. La quantification de l'énergie associée à ce mécanisme permettrait d'apprécier l'influence de celui-ci sur la phase post-rupture d'une avalanche rocheuse. Dans cet article, les distributions des tailles des blocs du massif rocheux et des débris sont présentées et comparées pour neuf cas d'avalanches rocheuses : cinq dans les montagnes Rocheuses canadiennes et quatre dans les Alpes européennes. Des degrés de fragmentation ont pu être estimés. Pour évaluer l'énergie de fragmentation, deux méthodes on été examinées : l'une est basée sur l'énergie de concassage et l'autre est basée sur l'énergie de sautage utilisée dans le domaine minier. Les résultats obtenus portent à croire qu'il y aurait une relation entre l'indice de réduction de taille (Rr = D50/d50) et l'énergie potentielle par unité de volume, normalisée par la résistance au double poinçonnement (?HG/?c). Les énergies de fragmentation calculées pour les neuf cas étudiés donne en moyenne 20 % de l'énergie potentielle. Une relation empirique entre Rr et ?HG/?c est proposée, et est par la suite utilisée pour définir un indice de désintégration (ID). Cet indice reflète la physique du processus de désintégration puisqu'il considère que l'indice de réduction de taille est fonction de l'énergie dissipée et de la résistance de la roche. Ces facteurs connus depuis longtemps n'avaient jamais été présentés d'une façon cohérente pour des cas d'avalanches rocheuses.Mots clés : avalanches rocheuses, désintégration, énergie de fragmentation, Rocheuses canadiennes, Alpes européennes.

Relationships of Scincid Lizards (Mabuya spp; Reptilia: Scincidae) from the Cape Verde Islands Based on Mitochondrial and Nuclear DNA Sequences

Partial DNA sequences from two mitochondrial (mt) and one nuclear gene (cytochrome b, 12S rRNA, and C-mos) were used to estimate the phylogenetic relationships among the six extant species of skinks endemic to the Cape Verde Archipelago. The species form a monophyletic unit, indicating a single colonization of the islands, probably from West Africa. Mabuya vaillanti and M. delalandii are sister taxa, as indicated by morphological characters. Mabuya fogoensis and M. stangeri are closely related, but the former is probably paraphyletic. Mabuya spinalis and M. salensis are also probably paraphyletic. Within species, samples from separate islands always form monophyletic groups. Some colonization events can be hypothesized, which are in line with the age of the islands. C-mos variation is concordant with the topology derived from mtDNA.

Phylogeography of Mabuya maculilabris (Reptilia) from São Tomé Island (Gulf of Guinea) inferred from mtDNA sequences

The pattern of genetic variation of the lizard Mabuya maculilabris from São Tomé Island (Gulf of Guinea) was investigated using a combination of three mitochondrial DNA gene fragments. Forty-eight haplotypes were recovered among 66 individuals covering the whole island. The genealogy inferred from the most parsimonious network of haplotypes allows us to detect two main and long branches departing from the putative group of oldest haplotypes. The tips of these branches exhibit star-like phylogenies, which may indicate of recently expanded populations, most probably from a small number of founders. A nested clade analysis suggests a complex pattern of past events that gave rise to the extant geographical pattern found in the haplotype distribution: past and allopatric fragmentation, range expansion, restricted gene Xow and long-distance dispersal. These results are consistent with the complex geological history of the island where important volcanic activity with extensive lava Xows has occurred during several periods. Mismatch- distribution analysis and AMOVA also support these conclusions. Substantial genetic structuring among these lizards was detected as well as high levels of diVerentiation between the southern edge populations (particularly those from the Rolas Islet) and the remaining ones. However, variation is low relative to the geological age of the island. Our results indicate that patterns of variation observed in reptiles in other oceanic islands are not indicative of those observed in the islands of the Gulf of Guinea.

A deep dig-hindsight on Holocene plant composition from ancient environmental DNA

Want a glimpse at past vegetation? Studying pollen and other plant remains, which are preserved for example in lake sediments or mires for thousands of years, allows us to document regional occurrences of plant species over radiocarbon-dated time series. Such vegetation reconstructions derived from optical analyses of fossil samples are inherently incomplete because they only comprise taxa that contribute sufficient amounts of pollen, spores, macrofossil or other evidences. To complement optical analyses for paleoecological inference, molecular markers applied to ancient DNA (aDNA) may help in disclosing information hitherto inaccessible to biologists. Parducci etal. (2013) targeted aDNA from sediment cores of two lakes in the Scandes Mountains with generic primers in a meta-barcoding approach. When compared to palynological records from the same cores, respective taxon lists show remarkable differences in their compositions, but also in quantitative representation and in taxonomic resolution similar to a previous study (JOrgensen etal. 2012). While not free of assumptions that need critical and robust testing, notably the question of possible contamination, this study provides thrilling prospects to improve our knowledge about past vegetation composition, but also other organismic groups, stored as a biological treasure in the ground.

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.

Heterobathmia pseuderiocrania Kristensen & Nielsen (Lepidoptera, Heterobathmiidae): identificación basada en DNA-barcoding y notas morfológicas e historia de vida de los estados inmaduros

Heterobathmia pseuderiocrania Kristensen & Nielsen (Lepidoptera, Heterobathmiidae): identification based on DNA-barcoding and notes on the morphology and life history of the immature stages. The larva morphology of the species Heterobathmia pseuderiocrania (Lepidoptera, Heterobathmiidae), a Nothofagus obliqua leafminer in Chile, is described. The tissue-feeding first and last instars are described. Also, the number of larval stages, some aspects of the biology and life cycle of the species are provided.

Direct visualization of supercoiled DNA molecules in solution.

The shape of supercoiled DNA molecules in solution is directly visualized by cryo-electron microscopy of vitrified samples. We observe that: (i) supercoiled DNA molecules in solution adopt an interwound rather than a toroidal form, (ii) the diameter of the interwound superhelix changes from about 12 nm to 4 nm upon addition of magnesium salt to the solution and (iii) the partition of the linking deficit between twist and writhe can be quantitatively determined for individual molecules.

Electron microscopy of RecA-DNA complexes: Two different states, their functional significance and relation to the solved crystal structure

Seven different electron microscopy techniques habe been employed to study the RecA protein of E. coli. This review provides a summary of the conclusions that have been drawn from these studies, and attempts to relate these observations to models for the role of RecA protein in homologous recombination.

Effect of early antiretroviral therapy during primary HIV-1 infection on cell-associated HIV-1 DNA and plasma HIV-1 RNA.

Background: Early initiation of combination antiretroviral therapy (ART) during primary HIV-1 infection may prevent the establishment of large viral reservoirs, possibly resulting in improved control of plasma viraemia rebound after ART cessation.Methods: Levels of cell-associated HIV-1 DNA and plasma HIV-1 RNA were measured longitudinally in 32 acutely and recently infected patients, who started ART <= 120 days after the estimated date of infection, and interrupted ART after 18 months (median) of continuous therapy. Averages of HIV-1 DNA and RNA concentrations present in blood 30-365 days after therapy interruption (median duration 300 days, range 195-358) were compared between patients who started ART <= 60 days after the estimated date of infection (early starters), those who started between 61 and 120 days (later starters), and, for HIV-1 RNA only, with 89 untreated participants of the Swiss HIV Cohort Study with documented sero-conversion and longitudinal measurements collected 90-455 days after the first positive HIV test.Results: In early ART starters, average levels of plasma HIV-1 RNA and cell-associated HIV-1 DNA after treatment interruption were 1 log(10) (P=0.008) and 0.4 log(10) (P=0.03) lower compared with later starters. Average post-treatment plasma HIV-1 RNA levels in early starters were significantly lower, respectively, compared with untreated controls (-1.2 log(10); P<0.0004).Conclusions: Early treatment initiation within 2 months after HIV infection compared with later therapy initiation resulted in reduced levels of plasma viraemia and proviral HIV-1 DNA for >= 1 year after subsequent ART cessation. Plasma HIV-1 RNA levels in early starters were also significantly lower than in untreated controls.