Effect of an Oxidative Guanine Lesion on Nucleosome Stability

During cellular respiration, reactive oxygen species (ROS) escape from mitochondria to oxidize cellular components including DNA. Of all four DNA bases, guanine has the lowest redox potential, resulting in the oxidation of DNA to form 8-oxoguanine (8-oxoG). Because it has an even lower redox potential than guanine itself, 8-oxoG is likely to be even further oxidized. Of the potential products that can result from the oxidation of 8-oxoG, the formation of spiroiminodihydantoin (Sp) is of particular interest, as it appears to be even more mutagenic than 8-oxoG. Previous investigators demonstrated that the Sp lesion significantly destabilizes duplex DNA when compared to both its parent lesion 8-oxoG and guanine. As the structure of genomic DNA in chromatin is more complex, our goal is to examine the effect of the Sp lesion on nucleosome formation. The Sp lesion is placed in specific locations along a 146-mer DNA oligonucleotide duplex, and this DNA is assembled onto chicken histones to form mononucleosomes by a double-dialysis method. Our data show that rapid nucleosome formation leads to kinetic trapping in non-equilibrium states. At equilibrium, the fraction of nucleosomes formed was the same for control and Sp-containing duplexes, regardless of lesion location or diastereomer. As the free energy of nucleosome formation was comparable despite the presence of the Sp lesion, we questioned whether the Sp lesion affects the rotational orientation of the proteins relative to the DNA. DNase I footprinting indicates distinctly different cleavage patterns both proximal to the lesion site and at locations as far as 30 base pairs away on the distal side of the core particle. We expect that additional footprinting experiments using diverse chemical reagents will allow us to more precisely define the positioning of the nucleosome on lesion-containing DNA.