Treacle and TOPBP1 control replication stress response in the nucleolus

Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress–response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle–TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle–TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.     

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.     

Characteristics of a Subthreshold Microwave Discharge in a Wave Beam in Air and the Efficiency of the Plasma-Chemical Reactor

Plasma Physics Reports - An analysis of a set of experiments on studying subthreshold microwave self/non-self-sustained (SNSS) discharges made it possible to estimate the volume of the active zone...     

Supplementary description and ontogenetic variability of Coryphaenoides affinis: rediscovery of the species after 145 years since its original description (Teleostei: Gadiformes: Macrouridae)

A large adult (138 mm head length) of the grenadier genus Coryphaenoides is described. It is identified as C. affinis hitherto known only from two juvenile type specimens collected by HMS Challenger off Uruguay in 1876. The diagnosis of the species is revised to include the discovery of considerable ontogenetic changes in squamation. Spinulation on the body scales are reduced or lost with size, while the armament of the head scales become amplified. The larger of the syntype specimens is designated as the lectotype. All three known specimens of C. affinis were collected in the southwestern Atlantic at depths between 3500 and 4000 m. A key for identification for the species of the subgenus Nematonurus is provided.     

A new powerful method for site-specific transgene stabilization based on chromosomal double-strand break repair

Transgenic insects are a promising tool in sterile insect techniques and population replacement strategies. Such transgenic insects can be created using nonautonomous transposons, which cannot be transferred without a transposase source. In biocontrol procedures where large numbers of insects are released, there is increased risk of transgene remobilization caused by external transposase sources that can alter the characteristics of the transgenic organisms lead horizontal transgene transfer to other species. Here we describe a novel, effective method for transgene stabilization based on the introduction of directed double-strand breaks (DSB) into a genome-integrated sequence and their subsequent repair by the single-strand annealing (SSA) pathway. Due to the construct's organization, the repair pathway is predictable, such that all transposon and marker sequences can be deleted, while preserving integration of exogenous DNA in the genome. The exceptional conservation of DNA repair pathways makes this method suitable for a broad range of organisms.     

Distribution of attention modulates salience signals in early visual cortex

Previous research has shown that the extent to which people spread attention across the visual field plays a crucial role in visual selection and the occurrence of bottom-up driven attentional capture. Consistent with previous findings, we show that when attention was diffusely distributed across the visual field while searching for a shape singleton, an irrelevant salient color singleton captured attention. However, while using the very same displays and task, no capture was observed when observers initially focused their attention at the center of the display. Using event-related fMRI, we examined the modulation of retinotopic activity related to attentional capture in early visual areas. Because the sensory display characteristics were identical in both conditions, we were able to isolate the brain activity associated with exogenous attentional capture. The results show that spreading of attention leads to increased bottom-up exogenous capture and increased activity in visual area V3 but not in V2 and V1.     

Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA

BACKGROUND: Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. PRINCIPAL FINDINGS/METHODOLOGY: We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. SIGNIFICANCE: Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.     

18 Possible extraterrestrial life: a quantum-chemical look on the silicon analogs of carbon biomolecules

The uniqueness of life on our planet has been an important topic of discussion in scientific literature for many decades. The most particular findings are in the fields of the structure of biomolecules and the mechanisms of their conformational and chemical transfers since they underlie all the biospheric processes of our planet. The compounds based on carbon are the subject of study of organic chemistry, which has an appropriate thoroughly developed classification of such substances; a number of approaches have been proposed for the analysis of composition and structure of the organic compounds, and a theoretical basis has been created, which describes the character of various chemical bonds involving carbon atoms. At the same time, since quite a while, there is a widely discussed hypothesis (Alison, 1968) concerning the possibility of existence of compounds, which are similar to organic, but are based on silicon atoms. Even in interstellar medium, among all the diversity of molecules detected, 84 are based on carbon, and 8 on silicon (Lazio, 2000), including four hybrid types, i.e. containing both silicon and carbon. According to approximate evaluations, the contents ratio of carbon to silicon in the space equals to 10:1, though the Earth’s crust consists of 87% of silicon in the form of oxides. In the Periodic Table, silicon is situated in the same group IV, like carbon. These two elements are largely similar in the structure of their valent electronic shells, and their noteworthy that previously it was stated (Lazio, 2000) that silicon-containing compounds are not as diverse in structure as carbon compounds. Despite having higher mass and radius, the atoms of silicon form double and triple covalent bonds (Wang et al., 2008). Therefore, the issue concerning the existence of silicon structures similar to carbon biomolecules, as well as the question of hypothetical “biochemical” processes involving non-carbonic analogs of aminoacids, carbohydrates, proteins, lipids, and other biomolecules, is still a matter of discussion in scientific and popular science literature. It is particularly notable that the modern methods of computational chemistry allow carrying out the estimating calculations of the structure and dynamics of such compounds, which is quite similar to the known approaches of substance modeling de novo in drug design. For instance, first by calculations (Nagase, Kudo, & Aoki, 1985), and later on experimentally (Abersfelder, White, Rzepa, & Scheschkewitz, 2010), aromaticity of cyclic carbohydrate-like derivatives of silicon was studied. In the present study, we used quantum-chemical semiempirical PM3 and ab initio B3LYP/6-311G(d,p) level of theory to investigate the peculiarities of several structural and thermodynamic parameters of molecules, which can be assumed as complete silicon analogs of carbonic L-amino acids and other biomolecules, so-called bricks of life: carbohydrates, nitrogenous bases, fatty acids, as well as vitamins and caffeine. The quantum-mechanical calculations that we made displayed that the molecules of silicon amino acids possess higher thermodynamic stability compared to carbon analogs. Thereby, silicon amino acids have a similar conformation freedom, increased values of dipole moment, as well as more pronounced electron-donor characteristics. Silicon analogs of carbohydrates, fatty acids, and nitrogenous bases are as well considered as heavier thermodynamically stable compounds, having special features in 3D-organization and worth further experimental study. The present work also deals with the question of the existence and stability of “alpha-helices” composed of silicon amino acids, because in the molecules of Si-analogs of aspartate and glutamate, we have discovered effective formation of intramolecular hydrogen bond (due to the side chain), which is highly important for Pauling–Corey alpha helix formation in natural L-amino acids (Kondratyev, Kabanov, & Komarov, 2010). Our estimations show that an “alpha helix” composed of 10 silicon alanine analogs is more stable in isolated state than a linear form of such macromolecule, which was not observed for a molecule of the same composition having a carbon backbone.     

The entropic nature of protein thermal stabilization

We performed thermodynamic analysis of temperature-induced unfolding of mesophilic and thermophilic proteins. It was shown that the variability in protein thermostability associated with pH-dependent unfolding or linked to the substitution of amino acid residues on the protein surface is evidence of the governing role of the entropy factor. Numerical values of conformational components in enthalpy, entropy and free energy which characterize protein unfolding in the “gas phase” were obtained. Based on the calculated absolute values of entropy and free energy, a model of protein unfolding is proposed in which the driving force is the conformational entropy of native protein, as an energy of the heat motion (T·S^NC) increasing with temperature and acting as an factor devaluating the energy of intramolecular weak bonds in the transition state.