Repair of damaged DNA plays a crucial role in maintaining genomic integrity and normal cell function. The base excision repair (BER) pathway is primarily responsible for removing modified nucleobases that would otherwise cause mutagenic consequences and lead to disease. The BER process is initiated by a DNA glycosylase through lesion recognition and excision. This work examines the initiation of BER within the context of packaged DNA. Using nucleosome core particle (NCP), the primary repeating unit of chromatin, as a model system, we characterize four glycosylases, human oxoguanine DNA glycosylase (OGG1), uracil DNA glycosylase (UDG), single-strand selective monofunctional uracil DNA glycosylase (SMUG1), and alkyladenine DNA glycosylase (AAG), repairing 8-oxo-7,8-dihydroguanine (8oxoG), uracil (U), and 1, N6-ethenoadenine (εA), respectively, each of which are representative lesions caused by oxidation, deamination and alkylation. We first evaluate OGG1 activity on lesions off the dyad axis in canonical NCPs using kinetic assay. We find that OGG1 can initiate BER at off-dyad positions in the absence of external cofactors and that this activity is facilitated by transient unwrapping of DNA from the histones. Using a DNA population with globally distributed U:G bp, we next investigate the influence of H2A variants on excision of U in NCP. We observe that the U with reduced solution accessibility are more readily excised in H2A.Z and macroH2A-containing NCPs than in canonical NCPs, reflecting the ability of these variants to facilitate excision at sites that are otherwise poorly repaired. We also identify a hexasome species within the macroH2A NCP ensemble that contributes to the high activities of both UDG and SMUG1 in the region close to the DNA terminus. These observations reveal potential functions for H2A variants in promoting BER and preventing mutagenesis within the context of chromatin. With the global fingerprint approach, we also obtain kinetic parameters of AAG at 49 positions throughout the NCP architecture. AAG activity is largely correlated with solution accessibility of εA and local histone architecture. Exceptions to solution accessibility dictating AAG activity reflects the impact of local nuances in NCP environment on glycosylase behavior.
Li, Chuxuan,
"Challenges for Base Excision Repair Enzymes: Acquiring Access to Damaged DNA in Chromatin"
(2020).
Chemistry Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.26300/bxhv-6557
