The luminescent characteristics of Ru(bpy) 2 dppz 2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine), a DNA light switch, were investigated in the presence of oligonucleotides containing single base mismatches or an abasic site. In water, the ruthenium luminescence is quenched, but, bound to well matched duplex DNA, the Ru complex luminesces. Here we show that with DNAs containing a defect, rac-, Δ-and Λ-Ru(bpy) 2 dppz 2+ exhibit significant luminescent enhancements above that with well matched DNA. In the presence of a single base mismatch, large luminescent enhancements are evident for the Δ-Ru isomer; the Λ-isomer shows particularly high luminescence bound to an oligonucleotide containing an abasic site. Similar increases are not evident with two common DNAbinding organic fluorophores, ethidium bromide and TO-PRO-3. Titrations with hairpin oligonucleotides containing a variable mismatch site show correlation between the level of luminescent enhancement and the thermodynamic destabilization associated with the mismatch. This correlation is reminiscent of that found earlier for a bulky rhodium complex that binds mismatched DNA sites through metalloinsertion, where the complex binds the DNA from the minor groove side, ejecting the mismatched bases into the major groove. Differential quenching studies with minor and major groove quenchers and time resolved emission studies support this metalloinsertion mode for the dppz complex at the defect site. Certainly these data underscore the utility of Ru(bpy) 2 dppz 2+ as a sensitive luminescent reporter of DNA and its defects.

It was recently shown that thymine dimers in the all-thymine oligonucleotide (dT)(18) are fully formed in <1 ps after ultraviolet excitation. The speed and low quantum yield of this reaction suggest that only a small fraction of the conformers of this structurally disordered oligonucleotide are in a position to react at the instant of photon absorption. In this work, we explore the hypothesis that conventional molecular dynamics simulations can be used to predict the yield of cyclobutane pyrimidine dimers in DNA. Conformations obtained from simulations of thymidylyl-(3'-5')-thymidine in various cosolvents were classified as dimerizable or nondimerizable depending on the distance between the C5-C6 double bonds of the adjacent thymine bases and the torsion angle between them. The quantum yield of cyclobutane pyrimidine dimer formation was calculated as the number of dimerizable conformations divided by the total number of conformations. The experimental quantum yields measured in the different solvents were satisfactorily reproduced using physically reasonable values for the two parameters. The mean dimerizable structure computed by averaging all of the dimerizable cis-syn conformations is structurally similar to the actual cis-syn dimer. Compared to the canonical B-form TT step, the most important structural property of a dimerizable conformation is its reduced helical twist angle of 22 degrees.

In all organisms, oxidation threatens the integrity of the genome. DNA-mediated charge transport (CT) may play an important role in the generation and repair of this oxidative damage. In studies involving long-range CT from intercalating Ru and Rh complexes to 5′-GG-3′ sites, we have examined the efficiency of CT as a function of distance, temperature, and the electronic coupling of metal oxidants bound to the base stack. Most striking is the shallow distance dependence and the sensitivity of DNA CT to how the metal complexes are stacked in the helix. Experiments with cyclopropylamine-modified bases have revealed that charge occupation occurs at all sites along the bridge. Using Ir complexes, we have seen that the process of DNA-mediated reduction is very similar to that of DNA-mediated oxidation. Studies involving metalloproteins have, furthermore, shown that their redox activity is DNA-dependent and can be DNA-mediated. Long range DNA-mediated CT can facilitate the oxidation of DNA-bound base excision repair proteins to initiate a redox-active search for DNA lesions. DNA CT can also activate the transcription factor SoxR, triggering a cellular response to oxidative stress. Indeed, these studies show that within the cell, redox-active proteins may utilize the same chemistry as that of synthetic metal complexes in vitro, and these proteins may harness DNA-mediated CT to reduce damage to the genome and regulate cellular processes.

Damage to genomic DNA leads to mutagenesis and disease. Repair of single base damage is initiated by DNA glycosylases, the first enzymes in the base excision repair pathway. Although eukaryotic packaging of chromosomal DNA in nucleosomes is known to decrease DNA glycosylase efficiency, the impact on individual glycosylases is unclear. Here, we present a model system in which we examine the repair of site-specific base damage in well-characterized nucleosome core particles by five different DNA glycosylases. We find that DNA glycosylase efficiency on nucleosome substrates depends not only on the geometric orientation of the damaged base but also on its identity, as well as on the size, structure, and mechanism of the glycosylase. We show via molecular modeling that inhibition of glycosylase activity is largely due to steric obstruction by the nucleosome core.