TY - JOUR

T1 - Concept of natural genome reconstruction. Part 4. Integration of extracellular double-stranded DNA fragments into the genome of hematopoietic stem cells and the formation of extrachromosomal intermediates

AU - Oshikhmina, S. G.

AU - Ruzanova, V. S.

AU - Ritter, G. S.

AU - Dolgova, E. V.

AU - Kirikovich, S. S.

AU - Levites, E. V.

AU - Efremov, Y. R.

AU - Karamysheva, T. V.

AU - Molodtseva, A. S.

AU - Raitsina, Y. V.

AU - Taranov, O. S.

AU - Sidorov, S. V.

AU - Nikonov, S. D.

AU - Leplina, O. Y.

AU - Ostanin, A. A.

AU - Chernykh, E. R.

AU - Kolchanov, N. A.

AU - Proskurina, A. S.

AU - Bogachev, S. S.

N1 - Concept of natural genome reconstruction. Part 4. Integration of extracellular double-stranded DNA fragments into the genome of hematopoietic stem cells and the formation of extrachromosomal intermediates / S. G. Oshikhmina, V. S. Ruzanova, G. S. Ritter [et al.] // Vavilov Journal of Genetics and Breeding. – 2026. – Vol. 30, No. 2. – P. 163-180. – DOI 10.18699/vjgb-26-18. – EDN UUEPTE. This work was supported by the Ministry of Science and Higher Education of the Russian Federation for the Institute of Cytology and Genetics (state budget-funded project No. FWNR-2026-0025) and by A.A. Purtov, I.N. Zaitseva and LLC “ES.LAB DIAGNOSTIC”.

PY - 2026

Y1 - 2026

N2 - To assess the possibility of integrating extracellular double-stranded DNA fragments into the recipient genome of hematopoietic stem cells, a complex substrate was constructed consisting of the entire M13F-AluI-M13R fragment and its two restrictive derivatives, appearing after hydrolysis with restriction endonucleases EcoRI and HindIII: M13FAluI-EcoRI and M13R-AluI-HindIII. The substrate contained a pBlueScript+ plasmid polylinker sequence, absent in the human genome, which framed the human AluI fragment cloned at the EcoRV site. Human bone marrow cells were treated with the DNA of the constructed complex substrate; taking into account the repair time of pangenomic single-strand breaks, preparations of metaphase plates were obtained. FISH revealed specific fluorescent signals. Simultaneously, DNA isolated from colonies obtained from bone marrow cells treated with a complex substrate was sequenced. Two rounds of sequencing were carried out: whole-genome and selective after targeted hybridization on metal beads. The results obtained indicate that homologous exchange between extrachromosomal and chromosomal DNA is possible. Integration into the genome via the single-strand annealing mechanism, involving microhomologies, is also possible. Intermediates were discovered that suggest the existence of an unusual integration into the genome at the nick of one end of the fragment and the other end of the fragment hanging freely into the interchromosomal space. A direct assessment of the possibility of integrating TAMRA-labeled fragments of fragmented human DNA and E. coli DNA into the genome of recipient cells was carried out using a human bone marrow cell model. The results obtained indicate that specific signals of homologous DNA are distributed throughout the chromosome body (human bone marrow cell model). Signals from nonhomologous E. coli DNA are predominantly concentrated in the centromeric regions of chromosomes. The ratio of the number of obtained reads with integration elements and FISH signals suggested the existence of a strong interaction between extracellular fragments and chromosomal DNA. Experiments have been conducted showing that linear plasmid DNA, after internalization into hematopoietic stem cells, forms a monomer ring. Internalized into the intracellular space, extracellular plasmid DNA is isolated together with chromosomal DNA after stringent purification and fractionation procedures. This fact suggests the existence of a strong ring associate of plasmid DNA and chromosome DNA formed without the participation of a protein framework in the form of a looped chromosomal strand.

AB - To assess the possibility of integrating extracellular double-stranded DNA fragments into the recipient genome of hematopoietic stem cells, a complex substrate was constructed consisting of the entire M13F-AluI-M13R fragment and its two restrictive derivatives, appearing after hydrolysis with restriction endonucleases EcoRI and HindIII: M13FAluI-EcoRI and M13R-AluI-HindIII. The substrate contained a pBlueScript+ plasmid polylinker sequence, absent in the human genome, which framed the human AluI fragment cloned at the EcoRV site. Human bone marrow cells were treated with the DNA of the constructed complex substrate; taking into account the repair time of pangenomic single-strand breaks, preparations of metaphase plates were obtained. FISH revealed specific fluorescent signals. Simultaneously, DNA isolated from colonies obtained from bone marrow cells treated with a complex substrate was sequenced. Two rounds of sequencing were carried out: whole-genome and selective after targeted hybridization on metal beads. The results obtained indicate that homologous exchange between extrachromosomal and chromosomal DNA is possible. Integration into the genome via the single-strand annealing mechanism, involving microhomologies, is also possible. Intermediates were discovered that suggest the existence of an unusual integration into the genome at the nick of one end of the fragment and the other end of the fragment hanging freely into the interchromosomal space. A direct assessment of the possibility of integrating TAMRA-labeled fragments of fragmented human DNA and E. coli DNA into the genome of recipient cells was carried out using a human bone marrow cell model. The results obtained indicate that specific signals of homologous DNA are distributed throughout the chromosome body (human bone marrow cell model). Signals from nonhomologous E. coli DNA are predominantly concentrated in the centromeric regions of chromosomes. The ratio of the number of obtained reads with integration elements and FISH signals suggested the existence of a strong interaction between extracellular fragments and chromosomal DNA. Experiments have been conducted showing that linear plasmid DNA, after internalization into hematopoietic stem cells, forms a monomer ring. Internalized into the intracellular space, extracellular plasmid DNA is isolated together with chromosomal DNA after stringent purification and fractionation procedures. This fact suggests the existence of a strong ring associate of plasmid DNA and chromosome DNA formed without the participation of a protein framework in the form of a looped chromosomal strand.

KW - FISH

KW - extrachromosomal ring structures

KW - integration into the genome

KW - whole genome sequencing

KW - FISH

KW - полногеномное секвенирование

KW - интеграция в геном

KW - экстрахромосомальные кольцевые структуры

UR - https://www.mendeley.com/catalogue/5743ab60-a61c-3270-9028-d38cda6c4779/

UR - https://elibrary.ru/item.asp?id=89192159

UR - https://www.scopus.com/pages/publications/105036309008

U2 - 10.18699/vjgb-26-18

DO - 10.18699/vjgb-26-18

M3 - Article

VL - 30

SP - 163

EP - 180

JO - Вавиловский журнал генетики и селекции

JF - Вавиловский журнал генетики и селекции

SN - 2500-0462

IS - 2

ER -