Graduation Date

Fall 12-16-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Interdisciplinary Graduate Program in Biomedical Sciences

First Advisor

Kishor K. Bhakat

Abstract

Human AP Endonuclease 1 (APE1) is the primary enzyme in the base excision repair (BER) pathway that repairs apurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome. Recently, through genome-wide mapping analysis, we have shown that APE1, as well as its post-translationally modified form, acetylated APE1 (AcAPE1), is enriched in the gene regulatory regions. The APE1/AcAPE1-enriched regions also harbor Guanine (G)-rich sequences that fold into DNA secondary structures called G-quadruplexes (G4s). Our lab has demonstrated a strong genome-wide correlation between the occurrence of G4 structures and the binding of APE1 and AcAPE1. However, it is unknown whether APE1/ AcAPE1 directly binds to the DNA G4 structures to regulate gene expression and DNA damage repair in the gene regulatory regions. This dissertation aims at characterizing APE1-G4 interaction and elucidating the role of this interaction in transcriptional regulation and DNA damage repair.

To characterize the APE1-G4 interaction and its role in transcriptional regulation of genes, particularly oncogenes, we investigated the G4-mediated expression of KRAS in Pancreatic ductal adenocarcinoma (PDAC) cell lines. Using different cell biology, biochemistry, and biophysical methodologies, we show that APE1 is a DNA G4-binding protein and regulates KRAS expression by controlling the loading of transcription factors on the KRAS promoter G4. Our findings unravel a new role of APE1 in regulating stable G4 formation and KRAS expression in PDAC cells. We also observed that AcAPE1 remains bound to the chromatin throughout the cell cycle, including all the phases of mitosis. We found that AcAPE1 colocalizes with G4 structures on the condensed mitotic chromatin. We demonstrate that APE1-G4 interaction is important for modulating DNA damage repair to facilitate post-mitotic transcriptional reactivation in daughter cells as knockdown of APE1 significantly reduced nascent RNA production.

Overall, our study has identified APE1 as a DNA G4-binding protein, highlighting the biological significance of APE1-G4 interaction in regulating transcription and endogenous DNA damage repair in gene regulatory regions.

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