Thymidine Glycol: The Effect on DNA Structure and DNA Binding by Site-Specific Proteins

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.