Numerous DNA transactions in eukaryotic nuclei are regulated by elements (enhancers) that can directly interact with their targets over large regions of DNA organized into chromatin. The mechanisms allowing communication over a distance in chromatin are unknown. We have established an experimental system allowing quantitative analysis of the impact of chromatin structure on distant transcriptional regulation. Assembly of relaxed or linear DNA templates into subsaturated chromatin results in a strong increase of the efficiency of distant enhancer–promoter E–P communication and activation of transcription. The effect is directly proportional to the efficiency of chromatin assembly and cannot be explained only by DNA compaction. Transcription activation on chromatin templates is enhancer- and activator-dependent, and must be accompanied by direct E–P interaction and formation of a chromatin loop. Previously we have shown that DNA supercoiling can strongly facilitate E–P communication on histone-free DNA. The effects of chromatin assembly and DNA supercoiling on the communication are quantitatively similar, but the efficiency of enhancer action in subsaturated chromatin does not depend on the level of unconstrained DNA supercoiling. Thus chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state characteristic for prokaryotic DNA.

Chromosomal translocations are a major cause of cancer. At the same time, the mechanisms that lead to specific chromosomal translocations that associate different gene regions remain largely unknown. Translocations are induced by double strand breaks (DSBs) in DNA. Here we review recent data on the mechanisms of generation, mobility and repair of DSBs and stress the importance of the nuclear organization in this process.

Design of selective anticancer drugs that are targeting RGD-binding integrin receptors which are known to be one of the perspective directions in the field of oncology. Significant progress in the development and application of these types compounds is already demonstrated. The accumulating body of basic and clinical evidence demonstrates potential significant effects on both in vitro and in vivo experimental models. However, the specific mechanism of action of these compounds is generally not a fully elucidated or the exact target responsible for the achievement of stated effects hasn't yet been defined sufficiently. To date eight types of integrin receptors, which are capable to recognize RGD-motif in natural ligands, has in fact been identified as (namely αIIbβ3, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1). Even so, the estimation of the affinity of one particular RGD-bearing anticancer agent is often based on the determination of the binding efficacy to only one or rarely two integrin receptors. Traditionally the range of targets is restricted by the integrins, which are known to be highly expressed in a particular model system. While potential interactions of such an agent with other RGD-recognizing receptors usually remain beyond the research. Nonetheless, such interactions may also affect the viability and behavior of cancer cells. In this review we attempt to critically analyze the principles of selectivity achievement in the case of RGD-bearing natural ligands and the applicability of these principles in the context of the anticancer drug design.

Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools.

— The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii)...

Triticain-α is a papain-like cysteine protease from wheat (Triticum aestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones. Meanwhile, its attachment to prodomain of the enzyme resulted in generation of insoluble (inclusion bodies) product that can be transformed into active protease upon refolding in vitro. The estimated yield of the product was affected by affinity six-histidine tag required for its single-step purification with the preferable N-terminal position of the tag. Expression of the two-domain Triticain-α construct in yeast (Pichia pastoris) strain GS115 and bacterial (Escherichia coli) strain Rosetta gami B (DE3) led to the accumulation of a soluble protein, which underwent autocatalytic maturation during expression (in yeast)/purification (in bacteria) procedures and exhibited pronounced protease activity. Furthermore, expression and solubility of such construct in Rosetta gami B (DE3) cells was improved by reducing the temperature of the bacterial growth yielding more active enzyme than yeast counterpart presumably due to facilitated formation of a characteristic disulfide bond critical for maintaining the catalytic site. We suggest that these findings are helpful for obtaining active Triticain-α preparations for scientific or medical applications, and can be employed for the design and production of beneficial recombinant products based on other papain-like cysteine proteases.