NMR-Based Structural Studies of Damaged DNAs

The structures of duplex DNAs containing damaged sites can be determined by the combined use of NMR and restrained molecular dynamics. The NMR of DNAs containing abasic sites can be challenging since there are two equilibrating forms of the abasic site present. The damaged sites also tend to have fewer NOE connectivities than conventional DNA. Nevertheless, sufficient experimental NMR data can be obtained on these samples and the procedures for refining the structures by restrained molecular dynamics are sufficiently robust for structures to be determined. Two case studies are presented. In one case it is shown that the presence of an abasic site gives rise to large-scale structural disruption when within a sequence that can give rise to DNA curvature. In the other case, a thymine glycol leads to structural changes primarily at, and adjacent to, the damaged site. Owing to the important biological consequences of damaged DNA, we have been investigating the struc- tures and dynamics of these systems for several years. In this paper, a progress report on the structural studies of two damaged DNAs is presented. The final, refined structures of these two damaged DNAs have not yet been obtained and the presentation here focuses on why these particular systems were chosen for study and the methods being used to determine the structures of the DNAs. The damaged sites that we are currently examining are thymine glycol, urea and abasic sites. Abasic sites are probably the most common form of damaged DNA since many repair pathways pass through the abasic site stage. Thymine glycol arises from the oxidation of thymine, which is more readily oxidized than adenosine, guanine or cytosine. Urea also arises from the oxidation of thymine. The structures of these three types of damaged sites are shown along with the structure of thymine. These damaged sites can be thought of as forming a series from thymine to the abasic site. Thymine glycol has an unsaturated ring that is unlikely to be able to stack into a DNA helix. Urea is much smaller than thymine glycol and has the capability to form some of the hydrogen bonding interactions of thymine, but will * Author to whom correspondence should be addressed. not have as favorable stacking interactions. The abasic site is the smallest of the series and has only the hydroxyl available for significant hydrogen bonding interactions. Since each of the three types of damaged DNA is biologically important, structural and dynamic information on each is needed to understand their effects. The comparison of the structural and dynamic results obtained on the members of this series may well allow an assessment of the relative importance of stack- ing, hydrogen bonding and steric effects on the struc- tures and dynamics of both damaged and undamaged DNAs. These damaged sites are being examined in different sequence and structural contexts since the biological effects of DNA damage are known to be dependent on sequence context. One sequence context being used is a normal, B-form DNA duplex for which we have already obtained a significant amount of data. The second sequence context is an A-tract DNA. Properly phased A-tracts can give rise to net curvature of DNA that can be important in the long range structure of DNA and gene regulation. In this paper the status reports on two structural determinations are given.