Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.     

Introgressive hybridization as a mechanism for species rescue

Rapid evolution on ecological time scales can play a key role in species responses to environmental change. One dynamic that has the potential to generate the diversity necessary for evolution rapid enough to allow response to sudden environmental shifts is introgressive hybridization. However, if distinct sub-species exist before an environmental shift, mechanisms that impede hybridization, such as assortative mating and hybrid inferiority, are likely to be present. Here we explore the theoretical potential for introgressive hybridization to play a role in response to environmental change. In particular, we incorporate assortative mating, hybrid inferiority, and demographic stochasticity into a two-locus, two-allele population genetic model of two interacting species where one locus identifies the species and the other determines how fitness depends on the changing environment. Simulation results indicate that moderately high values for the strength of assortative mating will allow enough hybridization events to outweigh demographic stochasticity but not so many that continued hybridization outweighs backcrossing and introgression. Successful introgressive hybridization also requires intermediate relative fitness at the allele negatively affected by environmental change such that hybrid survivorship outweighs demographic stochasticity but selection remains strong enough to affect the genetic dynamics. The potential for successful introgression instead of extinction with greater environmental change is larger with monogamous rather than promiscuous mating due to lower stochasticity in mating events. These results suggest species characteristics (e.g., intermediate assortative mating and mating systems with low variation in mating likelihood) which indicate a potential for rapid evolution in response to environmental change via introgressive hybridization.     

Strand invasion by mixed base PNAs and a PNA-peptide chimera

Peptide nucleic acid oligomers (PNAs) have a remarkable ability to invade duplex DNA at polypurine–polypyrimidine target sequences. Applications for PNAs in medicine and biotechnology would increase if the rules governing their hybridization to mixed base sequences were also clear. Here we describe hybridization of PNAs to mixed base sequences and demonstrate that simple chemical modifications can enhance recognition. Easily synthesized and readily soluble eight and 10 base PNAs bind to plasmid DNA at an inverted repeat that is likely to form a cruciform structure, providing convenient tags for creating PNA–plasmid complexes. PNAs also bind to mixed base sequences that cannot form cruciforms, suggesting that recognition is a general phenomenon. Rates of strand invasion are temperature dependent and can be enhanced by attaching PNAs to positively charged peptides. Our results support use of PNAs to access the information within duplex DNA and demonstrate that simple chemical modifications can make PNAs even more powerful agents for strand invasion. Simple strategies for enhancing strand invasion should facilitate the use of PNAs: (i) as biophysical probes of double-stranded DNA; (ii) to target promoters to control gene expression; and (iii) to direct sequence-specific mutagenesis.     

Plant-soil feedback as a mechanism of invasion by Carpobrotus edulis

Invasive plant species have been suggested to change the composition of the soil community in a way that results in a positive feedback for them and a negative feedback for the native plant community. Carpobrotus edulis, a species native to South Africa, is one of the most aggressive exotic species in Mediterranean Europe. Although several aspects of its invasion biology have been studied, the occurrence of plant-soil feedback has been scarcely investigated. We first checked for the existence of biotic resistance in soils from two invaded sites of Mediterranean Europe and one site in the native area. Secondly, we evaluated the effects of soil conditioning on the germination and plant growth of C. edulis and two key species of native dunes. Finally, we tested the effects of short- and long-term soil conditioning on the performance and reproductive effort of C. edulis. Our results show that at first there is a natural resistance to invasion by the soil biota. Later, biotic resistance in invaded soil is suppressed by the establishment of a soil community that enhances the growth of C. edulis and that negatively influences the growth and survival of the native plants. Long-term soil conditioning in the field resulted in shifts in the balance between vegetative growth and sexual reproduction. Long-term invasion was also reflected in high levels of endophyte colonization by chytrids in roots, although the physiological consequences of this colonization remain unknown. The results obtained illustrate a mechanism that explains how C. edulis breaks the initial biotic resistance of newly-invaded landscapes. Finally, this study highlights the importance of studying plant-soil interactions on different members of the plant community and temporal stages in order to fully understand invasion.     

Strand invasion by DNA-peptide conjugates and peptide nucleic acids.

Peptide nucleic acids (PNAs) and conjugates between oligonucleotides and cationic peptides possess superior potential for strand invasion at complementary sequences. We discovered that oligonucleotide-peptide conjugates and PNAs fall into three classes based on their hybridization efficiency; i) those complementary to inverted repeats within AT-rich region hybridize with highest efficiency; ii) those complementary to areas adjacent to inverted repeats or near AT-rich regions hybridize with moderate efficiency; and iii) those complementary to other regions do not detectably hybridize. The correlations between oligomer chemistry, DNA target sequence, and hybridization efficiency that we report here have important implications for the recognition of duplex DNA.     

Antidepressant-induced switch of beta 1-adrenoceptor trafficking as a mechanism for drug action

Reduction in surface beta(1)-adrenoceptor (beta1AR) density is thought to play a critical role in mediating the therapeutic long term effects of antidepressants. Since antidepressants are neither agonists nor antagonists for G protein-coupled receptors, receptor density must be regulated through processes independent of direct receptor activation. Endocytosis and recycling of the beta1AR fused to green fluorescent protein at its carboxyl-terminus (beta1AR-GFP) were analyzed by confocal fluorescence microscopy of live cells and complementary ligand binding studies. In stably transfected C6 glioblastoma cells, beta1AR-GFP displayed identical ligand-binding isotherms and adenylyl cyclase activation as native beta1AR. Upon exposure to isoproterenol, a fraction of beta1AR-GFP (10-15%) internalized rapidly and colocalized with endocytosed transferrin receptors in an early endosomal compartment in the perinuclear region. Chronic treatment with the tricyclic antidepressant desipramine (DMI) did not affect internalization characteristics of beta1AR-GFP when challenged with isoproterenol. However, internalized receptors were not able to recycle back to the cell surface in DMI-treated cells, whereas recycling of transferrin receptors was not affected. Endocytosed receptors were absent from structures that stained with fluorescently labeled dextran, and inhibition of lysosomal protease activity did not restore receptor recycling, indicating that beta1AR-GFP did not immediately enter the lysosomal compartment. The data suggest a new mode of drug action causing a "switch" of receptor fate from a fast recycling pathway to a slowly exchanging perinuclear compartment. Antidepressant-induced reduction of receptor surface expression may thus be caused by modulation of receptor trafficking routes.     

Periodic electric field as a biopolymer conformation switch: a possible mechanism

A theoretical model is proposed to describe the influence of a periodic electric field (PEF) upon a biopolymer. The biopolymer is treated as a classical mechanical system consisting of subsystems (molecular groups) which interact with each other through potential forces. The PEF is treated as a periodic driving force applied to a molecular group. The energy dissipation is considered using the model of fluid (viscous) friction. Arguments for the non-linear character of the friction-velocity dependence caused by the non-Newtonian rheology of a viscous medium are formulated.A forced molecular-group motion is investigated for the situation of a small driving-force period, with oscillations overdamped and a driving force consisting of more than one harmonic. As a result, it is established that the motion always gets to a terminal stage when only a small-scale vibration about some point, X ^*, takes place. The terminal motion is preceded by a transient characterized by the presence of a directional velocity component and so by a drift along a potential profile. the drift goes on until a barrier is met which has a sufficiently large steepness (the barrier height is not important). As a result, the point X ^* may happen to be remote from the conformation potential local minimum (conformational state). The physical reasons for the drift are described.If we consider the small-scale vibration about X ^* in the framework of the hierarchy of scales for intramolecular mobility, it can be regarded as an equilibrium mobility, whereas the drift can be regarded as a functionally important motion, and X ^* as a new conformational state which is realizable only in the presence of the PEF. It may be concluded, as the result of a consistent treatment and neglecting the small-scale vibration, that the conformational potential is modified by adding a linear term (in the one-dimensional case). In this connection, the set of conformational states can both deform (deviation of the positions of minima and their relative depth) and rearrange qualitatively (some minima can vanish and/or new minima can appear). In particular, the transition from one conformation to another one may happen due to rearrangement.     

A glutamine switch mechanism for nucleotide selectivity by phosphodiesterases

Phosphodiesterases (PDEs) comprise a family of enzymes that modulate the immune response, inflammation, and memory, among many other functions. There are three types of PDEs: cAMP-specific, cGMP-specific, and dual-specific. Here we describe the mechanism of nucleotide selectivity on the basis of high-resolution co-crystal structures of the cAMP-specific PDE4B and PDE4D with AMP, the cGMP-specific PDE5A with GMP, and the apo-structure of the dual-specific PDE1B. These structures show that an invariant glutamine functions as the key specificity determinant by a "glutamine switch" mechanism for recognizing the purine moiety in cAMP or cGMP. The surrounding residues anchor the glutamine residue in different orientations for cAMP and for cGMP. The PDE1B structure shows that in dual-specific PDEs a key histidine residue may enable the invariant glutamine to toggle between cAMP and cGMP. The structural understanding of nucleotide binding enables the design of new PDE inhibitors that may treat diseases in which cyclic nucleotides play a critical role.     

Induced structure of a helical switch as a mechanism to regulate enzymatic activity

Herpesviruses encode a protease that is activated by homodimerization at high enzyme concentrations during lytic replication. The homodimer contains two active sites, which are distal from the dimer interface. Assignment of backbone NMR resonances and engineering of a redox switch show that two helices position a loop containing catalytic residues within each active site.     

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.     

Allelopathy of a native grassland community as a potential mechanism of resistance against invasion by introduced plants

Successful plant invasions depend, at least partly, on interactions between introduced plants and native plant communities. While allelopathic effects of introduced invaders on native resident species have received much attention, the reverse, i.e. allelopathic effects of native residents on introduced plants, have been largely neglected. Therefore, we tested whether allelopathy of native plant communities decreases their invasibility to introduced plant species. In addition, we tested among the introduced species whether the invasive ones are more tolerant to allelopathy of native plant communities than the non-invasive ones. To test these hypotheses, we grew nine pairs of related (congeneric or confamilial) invasive and non-invasive introduced plant species (i.e. 18 species) in the presence or absence of a native grassland community, which consisted of three common forbs and three common grasses, with or without activated carbon in the soil. Activated carbon reduced the survival percentage and growth of introduced plants in the absence of the native plant community. However, its net effect on the introduced plants was neutral or even slightly positive in the presence of the native community. This might suggest that the native plant community imposed allelopathic effects on the introduced plants, and that these effects were neutralized or reduced by activated carbon. The invasive and non-invasive introduced plants, however, did not differ in their tolerance to such allelopathic effects of the native plant community. Thus, although allelopathy of native plant communities might increase their resistance against introduced plants, there was no evidence that tolerance to allelopathy of native plant communities contributes to the degree of invasiveness of introduced plants.