Abstract
Thymidine glycol (5,6-dihydroxy-5,6-dihydrothymidine, Tg) is a major type of oxidative damage in DNA. During chemical oligonucleotide synthesis, Tg residue was incorporated in the different positions of 17 b.p. DNA duplexes, which differ in one base pair in the internal part. According to UV-melting curves, Tg destabilizes the double helix in a sequence independent manner. In contrast, the localized alterations in duplex structure were shown by CD spectroscopy to depend on the type of base pairs flanking the Tg lesion. Molecular dynamics simulations demonstrate that Tg is partially out of the double helix. For the first time, Tg impact on several site-specific DNA-binding proteins is studied, namely p50 and p65 subunits of nuclear factor kappa-B (NF-κB) and DNA methyltransferase SsoII (M.SsoII). Our results show that p50/p50 and p65/p65 homodimers of NF-κB can tolerate a single Tg residue in the binding site quite well. Nevertheless the homodimers have different affinities to the oxidized κB site depending on the Tg position. M.SsoII can act as a transcription repressor when bound to the regulatory site. M.SsoII demonstrates decreased affinity and lowered methylation efficiency when its methylation site contains Tg in the central position. Single Tg in one half of the regulatory site decreases M.SsoII affinity to the oxidized DNA, whereas Tg presence in both half-sites prevents M.SsoII binding to such ligand.
Highlights
It is widely established that it is essential to understand the main features of biopolymer interactions in order to develop methods for efficient monitoring the biological activities, as well as the reasons of their changes
Since Tg is labile under alkaline conditions, we applied a short cycle of ammonia treatment at room temperature for oligonucleotide deprotection
The oligonucleotide mixtures after the melting procedure were analyzed by reverse-phase HPLC in order to determine the integrity of the DNA strands
Summary
It is widely established that it is essential to understand the main features of biopolymer interactions in order to develop methods for efficient monitoring the biological activities, as well as the reasons of their changes. Thymidine glycol (5,6-dihydroxy-5,6-dihydrothymidine, Tg) is a major type of pyrimidine residue oxidative damage in DNA. It is formed as a result of thymine base oxidation by genotoxic factors such as ultraviolet or ionizing radiation and chemical oxidants [1]-[3]. This can be a result of aerobic metabolism [4]. Tg can be used as a genetic biomarker of oxidative DNA damage along with other biomarkers such as 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) [7]-[9]. There are four diastereomers of Tg because it contains two chiral carbon atoms, but Tg exists in solution as either the 5R cis-trans pair (5R,6S; 5R,6R) or the 5S cis-trans pair (5S,6R; 5S,6S) due to epimerization at the C6 position [10]
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