Formation of the D-loop structure complexes between DNA and oligonucleotides and affinity modyfication of DNA by chemically reactive derivatives of oligonucleotides lead to the recombination
Olga I Sinitsina, Konstantin V. Stoletov, Dmitry V. Bugreev, Galiya A. Maksakova, Irina F. Bugreeva, Elena A. Vasyunina, Georgy A. Nevinsky
Med Sci Monit 2000; 6(4): BR653-657
Background: Affinity modification of DNA by chemically reactive derivatives of complementary oligonucleotides (ODNs) and antisense ODNs has shown an application for the inhibition of gene expression and the growth of viruses and parasites in high organisms. Unfortunately, the rapid advancement of antisense therapeutic approaches is not parallel to the investigation of possible consequences of antisense and gene-directed ODNs on genetic material of the cells being treated. Here we tried for the first time to estimate a possible genetic impact of antisense ODNs and their chemically reactive derivatives on the cells using bacteria and the plasmid DNA.
Material/Methods: Recombination of direct repeats, induced by the formation of reversible complexes of plasmid DNA with complementary ODNs and after covalent binding of the alkylating derivative of the ODNs with DNA, has been investigated. For this purpose, a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR 327. This plasmid was used to construct DNA containing AT- and GC- rich sequences placed in the central region of the polylinker.
Results: Transformation of E. coli cells with the plasmids (and with mixtures of the plasmids with d(pN)17, complementary to the AT- and GC- rich sequences) did not produce deletions. After modification of plasmids with alkylating derivatives of d(pN)17,the deletion of the polylinker DNA region (recombination) revealed the restoration of the tet gene function. The same effect was found at the cell transformations with the D-loop complex of the plasmids with ODNs, but the frequency of the transformants was about 1.5-2 times lower. The data obtained demonstrate that the complexes of DNA with complementary ODNs and the modification of the plasmids by reactive ODN derivatives result in induction of the recombination process and in loss of genetic material.
Keywords: Bacteria, recombination, repair, plasmid DNA modification, alkylating oligonucleotide derivatives