Oxidation of DNA by Long-Range Charge Transport

Citation

Nuñez, Megan Elizabeth (2002) Oxidation of DNA by Long-Range Charge Transport. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ZQ5T-9Z15. https://resolver.caltech.edu/CaltechTHESIS:01312012-114925650

Abstract

Ever since the double helical structure of DNA was elucidated, it has been proposed that charge might move through the stacked base pairs of the double helix because of the electronic coupling of the π orbitals of the nucleotide bases with neighboring bases. Here it is demonstrated that electronic "holes" generated by a one-electron oxidation of DNA can result in permanent lesions on guanine bases up to 200 Å away from the intercalating oxidant as a result of such charge migration. Both rhodium and ruthenium complexes, covalently tethered to the 5' end of a double-stranded oligonucleotide and intercalated into the base stack, can with photoactivation promote oxidation of guanines in 5'-GG-3' sites over this distance. Since charges can move efficiently through the DNA oligonucleotides, it was important to characterize this reaction in more detail, and to extend observations of charge transport through DNA to larger and more complicated DNA assemblies that more closely mimic its structure in vivo.

Long-range oxidative damage to guanine doublets in DNA is shown to compete for oxidation with other reactions, such as the repair of thymine dimers. When both thymine dimer lesions and guanine doublets are present, both can be oxidized by a photoexcited rhodium complex, although each in lower yield than in the absence of the other. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Therefore electronic "holes" generated on genomic DNA might not of necessity cause DNA damage, but could also be funneled onto proteins or other oxidizible sites.

Using a variety of intercalating photooxidants targeted to a specific site on a restriction fragment by an appended triplex-forming oligonucleotide, the upper distance limits and sequence effects on long-range charge transfer through DNA were examined. Charge migration occurs in both directions from the intercalator and on both DNA strands of the target, but the oxidation is significantly more efficient to the 3' side of the triplex, over 25-38 base pairs. When intercalators were tethered directly to the 5' terminus of the triplex-forming strand as opposed to the center, significant amounts of oxidative damage was generated only in the immediate vicinity of the intercalation site, suggesting that the base stack is distorted at the 5' end of the triplex region in the duplex/triplex junction. Targeting of photooxidative damage by triplex formation extends previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed.

Within eukaryotic cells most DNA is packaged as nucleosome core particles, made up of ~146 base pairs of DNA wrapped around a core of histone proteins. Photoexcited rhodium complexes were also used to explore charge transport through DNA within these structures. Although histone proteins inhibit intercalation of a noncovalent rhodium complex, they do not prevent oxidation of 5'-GG-3' sites, the signature of oxidative charge transport through DNA. Furthermore, some of these sites are not directly accessible to a solution-bound oxidant due to his tones in the major groove, and thus they must be oxidized from a distance. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage from solution-borne molecules, it does not protect the DNA from charge transfer damage through the base pair stack. In support of this assertion, guanine bases within nucleosomal DNA were oxidized at a distance of over 23 base pairs from a covalently-tethered rhodium intercalator.

The environment within the cell nucleus contains a variety of other proteins and small molecules that could potentially influence the migration of charge through DNA. Using the rhodium photochemistry, the oxidation of guanine by photoexcited rhodium complexes inside of nuclei from cultured human cells was examined and compared with the oxidative damage on bare genomic DNA. Oxidation occurs preferentially at the 5'-guanine of 5'-GG-3' sites, indicative of base damage by DNA-mediated charge transport chemistry. Moreover, oxidative damage occurs at protein-bound sites which are inaccessible to rhodium. Thus, on transcriptionally active DNA within the cell nucleus, DNA-mediated charge transport acts to induce base damage from a distance. Direct interaction of an oxidant is not necessary to generate a base lesion at a specific site within the nucleus.

All of these observations indicate that charges can migrate along DNA within the cell. These observations require a reconsideration of cellular mechanisms for DNA damage and repair, and present new avenues for exploration in the design of DNA-based drugs and therapies.

Thesis Files