The spread of antibiotic-resistant bacterial strains is becoming a serious problem in modern healthcare. The causative agent of tuberculosis Mycobacterium tuberculosis (Mtb) causes the largest number of deaths among bacteria. Mtb has multidrug and extensively drug resistance. For mutations to occur in the genome of a pathogen, it is necessary to understand the fundamental foundations of their occurrence, namely the processes of replication, recombination and repair. The replicative apparatus of bacteria, in a strictly allotted time, must produce a copy of the genomic DNA. Mutations occur during cell division. Such mutations are eliminated by DNA repair enzymes of mismatch repair system. This project proposes to study the biochemical activity of DNA repair enzymes involved in the elimination of mismatched nucleotides.

Relevance

To date, the DNA repair system in mycobacteria remains poorly understood. Despite the fact that during the sequencing of the M. tuberculosis genome, homologues of genes encoding enzymes involved in the base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining pathways (NHEJ), etc., have been found, the substrate specificity of many enzymes still remains unexplored.

The knowledge gained during experiments on the Escherichia coli model organism underlies most of the research on DNA repair in bacteria. The functions of a number of enzymes and new DNA repair enzymes previously undetected in prokaryotic cells, as well as the number of DNA repair pathways, differ significantly in the case of Mycobacterium tuberculosis. Moreover, the mycobacterium genome does not contain homologues of known genes of the DNA repair mechanism.

Objective

The aim of the project is the biochemical characterization of previously unexplored DNA repair enzymes.

Expected results

The problem of tuberculosis is very serious both in the world and in Kazakhstan. The causative agent of tuberculosis has become resistant to available antibiotics. The results of this project have the prerequisites to identify the role of DNA repair enzymes in the emergence of antibiotic resistance. In addition, DNA repair enzymes represent promising new targets for the development of next-generation antibiotics.

Principal investigator

Abeldenov S.K. H-index – 3; ResearcherID F-5139-2015; ORCID 0000-0002-6974-9138; Scopus Author ID 56674705400

Members of the research group

Turgimbayeva A.M. H-index – 1; ResearcherID N-6857-2017; ORCID 0000-0001-7263-1643; Scopus Author ID 57202383621

Kirillov S.O. H-index – 1; ResearcherID N-6322-2017; ORCID 0000-0001-6229-5762

Zein U.O.          

Publications and security documents of the project manager and members of the research group

1. Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay. Amanzholova M., Shaizadinova A., Bulashev A., Abeldenov S. Vet Res Commun. ‒ 2023. DOI: 10.1007/s11259-023-10212-z
2. Rapid and highly sensitive LAMP-CRISPR/Cas12a-based identification of bovine mastitis milk samples contaminated by Escherichia coli. Shaizadinova A., Amanzholova M., Kirillov S., Bulashev A., Abeldenov S. Journal of Agriculture and Food Research. ‒ 2023, DOI: 10.1016/j.jafr.2023.100721
3. Cloning and characterization of the major AP endonuclease from Staphylococcus aureus. Turgimbayeva A, Zein U, Zharkov DO, Ramankulov Y, Saparbaev M, Abeldenov S. DNA Repair (Amst). 2022
4. Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori. Turgimbayeva A, Abeldenov S, Zharkov DO, Ishchenko AA, Ramankulov Y, Saparbaev M, Khassenov B. PLoS One. 2018 Aug 15;13(8):e0202232. doi: 10.1371/journal.pone.0202232.
5. Obtaining and characterization of monoclonal antibodies against recombinant extracellular domain of human epidermal growth factor receptor 2. Sarina N, Abeldenov S, Turgimbayeva A, Zhylkibayev A, Ramankulov Y, Khassenov B, Eskendirova S. Hum Antibodies. 2018 Feb 5;26(2):103-111. doi: 10.3233/HAB-170327.
6. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis. Abeldenov S, Talhaoui I, Zharkov DO, Ishchenko AA, Ramanculov E, Saparbaev M, Khassenov B. DNA Repair (Amst). 2015 Sep;33:1-16. doi: 10.1016/j.dnarep.2015.05.007.

Results achieved

2023

The genes of DNA repair enzymes were cloned into expression vectors to obtain various forms of recombinant proteins. The obtained recombinant vectors were sequenced. The expression of recombinant proteins was optimized (selection of producer strains, selection of conditions for temperature and time of biomass cultivation, concentration of the inductor). The development of cellular biomass, lysis and chromatographic purification of recombinant proteins using various chromatography methods to obtain recombinant proteins of high purity purification was carried out.

2024

Duplex DNA substrates were prepared, for example: U-22-(5′-TAMRA-d(CACTTCGGAUTGTGACTGATCC)), 22-mer, where U=2′deoxyuridine in position 10, complementary oligonucleotide 22-COMPL-G (5′-GGATCAGTCACAGTCCGAAGTG). Analysis of the cleavage of duplex DNA substrates showed the presence of enzyme activity towards the substrate containing uracil in the non-complementary position. As a result of optimization, the buffer composition was determined: 25 mM Hepes-NaOH pH 6.5, 2.5 mM MgCl2, 5 mM DTT, 0.1 mg/ml BSA, 0.1% Triton-X. When determining the dependence on divalent metal cations, it was found that 2.5 mM MgCl2 in the reaction mixture promoted catalysis. The cleavage reaction using a substrate containing uracil showed that the maximum activity was observed using 1.4 μM enzyme concentration at 20 nM substrate concentration. The substrate cleavage site was determined at the location of the mismatched base. It was found that using 2 μM beta-clamp concentration increased substrate processing by 20-30% compared to the control. Using databases, known amino acid sequences of beta-clamp motifs of protein partners were identified and compared to determine conserved regions involved in protein-protein interactions. With respect to the sequence of the enzyme under study, a conserved region was found that has the sequence EYRLF.