Graduation Date

Summer 8-9-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Kishor K. Bhakat

Abstract

Apurinic/apyrimidinic (AP) sites are the most frequently formed DNA lesions in the genome. The primary enzyme to repair AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the base excision repair (BER) pathway. Mammalian APE1 has a unique N-terminal unstructured tail and has both DNA repair and transcriptional regulatory activities. Our lab discovered that APE1 can be regulated via post-translational acetylation of lysine residues 6, 7, 27, 31, and 32. The role of mammalian APE1 in repair has been extensively studied and well characterized. However, the regulatory role of APE1 acetylation (AcAPE1) in the context of both DNA damage repair and transcriptional regulation has not been elucidated.

We show that APE1 is acetylated after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable Lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of positive charges of Lysine residues in the N-terminal domain of APE1 induces a conformation change; this, in turn, enhances the AP-endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity.

By mapping genome-wide occurrence of endogenous AP site damages and binding of repair proteins APE1 and AcAPE1, we demonstrate that oxidative base damages predominantly occur in transcriptionally active regions, particularly G-quadruplex (G4) sequences and activation of APE1-mediated BER pathway promotes the formation of G4 structures in the genome. Loss of APE1 or its acetylation abrogates the formation of G4 structures in cells. Acetylation of APE1 enhances its residence time and facilitates transcription factor loading, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels an acquired function of endogenous base damage and AcAPE1-mediated BER in regulating transcription.

Together this study highlights role of AcAPE1 in coordinating potential functional overlap between DNA damage repair activity and transcriptional regulation.

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