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Primary Wellesley Thesis Advisor
DNA constantly endures oxidative damage from a variety of sources, including ultraviolet radiation, heavy metal exposure, and reactive oxygen species that escape from the mitochondria during cellular respiration. The latter cause is particularly pernicious, as its endogenous nature makes it unavoidable. Consequently, oxidative lesions continually form within DNA and must be repaired to prevent mutagenesis. Guanine is the most commonly oxidized base, due to its low redox potential, and forms the 8oxoguanine (8oxoG) lesion. 8oxoG has an even lower redox potential than guanine itself and so may be further oxidized to form a variety of secondary products, including the spiroiminodihydantoin (Sp) lesion. Sp can mispair with either guanine or adenine, resulting in GàC and GàT transversion mutations. This mutagenicity, coupled with prior determinations of negative effects on duplex stability, prompted us to consider how Sp might affect nucleosome assembly and stability.
We utilized polyacrylamide gel electrophoresis (PAGE) to examine the stability of nucleosomes assembled via dialysis and found that the presence of Sp only marginally affected nucleosome stability, despite destabilization of the DNA duplex. Furthermore, Sp imposed a significant difference between nucleosomes assembled through a fast vs. slow dialysis, implying the potential for kinetic trapping. When we further examined these nucleosomes through DNase I footprinting, we discovered that Sp significantly altered the rotational and translational positioning within the nucleosome core particle. This finding both offers insight into how nucleosomes may accommodate the bulky lesion and carries far-reaching implications for gene regulation.
Finally, we developed and optimized a system for studying the pathway and kinetics of nucleosome assembly in the presence of Sp through Förster Resonance Energy Transfer (FRET). We placed one fluorophore on the DNA duplex and the other on the histone octamer, thereby coupling the distance dependence of FRET to the nucleosome assembly process. We intended to induce nucleosome assembly and disassembly through salt titration, ultimately comparing the data taken with and without Sp to elucidate the lesion’s effects. While to date we are still working to achieve a clear contrast between the signal from assembled and disassembled nucleosomes, we anticipate that we will soon be able to proceed with the bulk of our kinetics study.
Available for download on Wednesday, April 20, 2022