Oxidative DNA damage is the fundamental molecular cause of aging, neurodegeneration, and many types of cancer. Single DNA base lesions are repaired via the base excision repair (BER) pathway, a coordinated series of enzyme-catalyzed chemical reactions in which the damaged base is removed and replaced. Despite the importance of this process, little is known about the kinetics of BER on nucleosomes, DNA-protein complexes that make up the majority of eukaryotic genomic DNA. In the proposed research, the kinetics of each step in the BER pathway will be examined as a function of the position of the lesion within the nucleosome.
Specific aims i nclude: i) determining the rate of removal of 8-oxo-7,8-dihydroguanine (8-oxoG), the most prevalent oxidative lesion in humans, from nucleosomal DNA by the glycosylase hOGG1; ii) measuring the rate of downstream processes in BER, including deoxyribophosphate backbone hydrolysis by apurinic/apyrimidinic endonuclease 1, DNA extension by polymerase b, and DNA backbone nick sealing by ligase III-a; and iii) examining the effects of epigenetic changes, such as post-translational modification, binding by gene silencing factors, and action by chromatin remodeling complexes, on the rate of BER. These experiments will be carried out on a model system employing the Widom 601 DNA sequence, a 145-mer duplex that forms a homogeneous population of nucleosomes. Reaction rates will be determined by fitting time course data to kinetic models. Through the course of this research, training and experience will be provided in the areas of DNA synthesis, protein expression and purification, gel electrophoresis, DNA cleavage assays, radiolabeling, and enzyme kinetics.
Ineffective repair of DNA damage can lead to aging, cancer, and disease. In human cells, DNA damage repair is complicated by the fact that DNA is packaged as chromatin. This research will focus on understanding the specific chemical mechanisms by which DNA damage is repaired in the chromatin environment.
| Olmon, Eric D; Delaney, Sarah (2017) Differential Ability of Five DNA Glycosylases to Recognize and Repair Damage on Nucleosomal DNA. ACS Chem Biol 12:692-701 |
