Organic radicals and chromatin
Despite the proposal that the anticancer and antibiotic activity of the enediyne anticancer antibiotics is the result of the radical-mediated oxidative cleavage of DNA, few studies of the reactivity of organic radicals have utilized intact chromatin to emulate the environment of DNA in eukaryotic cells. Furthermore, although simple alkyl and aryl radicals have been implicated in the carcinogenicity of a number of chemical species, the published studies on their reactivity have focused primarily on purified nucleic acids. Thus, little is known about how the incorporation of DNA and histones into the larger chromatin assembly affects the reactivity of these biomolecules with simple organic radicals.
Textbook representation of how DNA is packaged in cells (from Alberts):
Chromatin structure models
Aside from addressing this fundamental issue, the investigation of the reactivity of organic radicals with chromatin will also be used to probe chromatin structure. The structure of the basic chromosomal building block, the nucleosome, has been determined (below); however, many key questions related to higher order chromatin structure have not been resolved. For example, what is the position of H1 on the nucleosome? How do nucleosomes pack to form chromatin fibers? What influence does histone tail phosphorylation exert on the interaction of the tails with DNA and on overall chromatin structure?
Figure 1. X-ray crystal structure of the nucleosome core particle from Luger, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J., "Crystal structure of the nucleosome core particle at 2.8 Angstrom resolution," Nature 1997, 389, 251260.
The long-term goal of this research is to elucidate, at the molecular level, the structural features of nucleosome packing in chromatin and to determine how this structure changes during cellular growth and development. The first step in pursuit of this goal is to map static chromatin structure by determining the location and nature of the modification of chromatin components by organic radicals. This idea arises from the hypothesis that the identification of the solvent-accessible surfaces of the biomolecules in these large assemblies, coupled with what is already known about nucleosomal structure, will lead to a molecular level picture of nucleosome packing in the 30 nm chromatin fiber (three proposed models for which are shown below). Once a versatile system for footprinting such large assemblies of biomolecules has been developed (and once their structures are known), structural changes in chromatin in other cellular processes will be probed.
Our initial work in this area has focused on the effects of photogenerated methyl radical on complexes of DNA with the linker histone H1. In a completely unexpected and novel outcome, we found that methyl radicals caused dissociation of H1 from the DNA, suggesting an additional mode of action for the carcinogenic behavior of carbon-centered radical and for the anticancer activity of the enediynes. We have determined that this disssociation is caused by conversion of the lysine side chains (the ammonium groups of which are attracted to the phosphate groups in DNA) to aldehydes (which are not attracted to DNA). This finding is the basis of further studies aimed at using these reagents to label proteins (see the next research area page).
Our future work will include footprinting (via DNA cleavage) the DNA in a nucleosome and in chromatin composed of 4 to 22 nucleosomes to determine which of the above models is correct for nucleosome packing.