Vol 59, No 4 (2025)

The review describes methods for correcting errors in synthetic oligonucleotides and genetic constructs derived from them. Methods for isolating oligonucleotides with perfect structure from oligonucleotide pools are discussed. Methods for correcting errors in DNA structure using mismatch–specific endonucleases and proteins of the bacterial DNA repair system are considered. Examples of practical application of the eveloped methods for adjusting the structure of synthesized genetic constructs are given.

The development of resistance to trastuzumab in HER2-positive breast cancer is a serious clinical problem that limits the effectiveness of targeted therapy. In a significant proportion of patients, the mechanisms of resistance development remain poorly understood. The BT-474 cell line was selected as an optimal model for the study because it represents a HER2-positive luminal B subtype breast cancer cell line. To identify molecular mechanisms of resistance, a comprehensive transcriptomic analysis based on RNA-seq data comparison of three independent datasets including both sensitive and trastuzumab-resistant variants was applied. The methodological approach included multi-step bioinformatics analysis followed by identification of regulatory interactions. The study identified genes with increased expression (FUCA2, HSPE1, SHLD1, NMD3) and genes with decreased expression (GPC5, FSTL1, ATG16L2, POLD2) in resistant cells. Key transcription factors (E2F1, MYC, YBX1, HEY1, NFIC, TFAP2A, AP-1/JUN, NCOA1) regulating the expression of the detected genes during resistance development were identified. The identified changes indicate a complex reprogramming of transcriptional activity affecting cell cycle processes, DNA repair, metabolism, and epithelial-mesenchymal transition. The findings expand the understanding of the molecular mechanisms of trastuzumab resistance and open prospects for the development of novel therapeutic strategies to overcome drug resistance in HER2-positive breast cancer.

Staphylococcus aureus Era GTPase is one of the ribosome maturation factors. Era GTPase is involved in the late stages of small (30S) ribosome subunit assembly and regulates formation of the entire 70S ribosome. Study of the structure of the Era and 30S subunit complex from S. aureus will help in studying the processes of ribosome maturation and mechanisms of protein synthesis regulation in this pathogenic microorganism. This paper presents protocols for obtaining Era GTPase and immature 30S ribosome subunits from S. aureus, as well as a protocol for assembling their complex for further structural studies.

The unconventional halotolerant yeast Debaryomyces hansenii is of great importance in biotechnology and food industry, and in basic research it serves as a model for studying the molecular mechanisms of resistance to increased salinity and osmotic pressure. We have previously established an efficient method for editing the D. hansenii genome using the CRISPR/Cas9 system. In turn, this has stimulated further investigation of the structure and physiological role of DNA double-strand break repair pathways in D. hansenii. The aim of the present work was to evaluate the involvement of key components of the DNA double-stranded break repair system by the non-homologous end joining (NHEJ) mechanism in the resistance of D. hansenii to DNA-damaging compounds and compounds that induce oxidative, high salinity and osmotic stress. Using the CRISPR/Cas9 system, mutant strains with knockout of the DEHA2F10208g (DhKU70), DEHA2B01584g (DhKU80), and DEHA2G04224g (DhLIG4) genes encoding key components of NHEJ were obtained. It was found that mutant strains, unlike the wild-type strain, are sensitive to chemical compounds that damage DNA, as well as to compounds that cause oxidative stress. Osmotic and high salinity stresses and vanillin do not cause significant changes in the rate of colony formation of mutant strains. Unexpectedly, mutant strains exhibit increased resistance to caffeine compared to the wild-type strain. The data indicate that the NHEJ systems of D. hansenii play a significant role in the response to DNA-damaging and oxidative types of stress. The importance of the NHEJ system in the processes of maintaining yeast cell homeostasis should be taken into account when creating strains producing valuable substances.

Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. It has been suggested that the key factors determining the positioning of ORC in the genome are DNA-binding proteins that form various DNA regulatory elements, including insulators, promoters, and enhancers, thereby linking the replication program to different levels of transcriptional regulation. Previously, we demonstrated that the Su(Hw) protein is the first example of such a protein. Subsequent studies identified a number of other DNA-binding proteins, including CG10543, which may be responsible for the formation of corresponding regulatory elements and the recruitment of transcriptional and replication complexes to their binding sites. It has been shown that the Drosophila CG10543 protein interacts with the deubiquitinating (DUB) module of the SAGA complex. The binding sites of the CG10543 protein are predominantly located in the promoter regions of active genes and colocalize with the SAGA and dSWI/SNF chromatin modification and remodeling complexes, as well as with the ORC replication complex. To investigate the role of the CG10543 protein in transcriptional regulation, an RNA-Seq experiment was conducted in Drosophila S2 cells under normal conditions and upon RNA interference of the CG10543 protein. It was shown that the CG10543 protein affects the transcription of 469 genes, with a significant portion of these genes (23%) being ecdysone-dependent genes. Ecdysone is the main steroid hormone in Drosophila, responsible for Drosophila metamorphosis and has a significant effect on the expression of many genes during development. We demonstrated that CG10543 sites colocalize with the CBP protein and the histone mark H3K27Ac, which are characteristic of active regulatory elements. The CG10543 protein also colocalizes with the CP190 protein, suggesting a potential mechanism of transcriptional regulation through the formation of long-range interactions between regulatory elements.