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Updated: Sep 30, 2025
A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1
Published on: March 18, 2017
The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in
Suravi Pramanik1, Yingling Chen1, Heyu Song1
1Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Abstract:
Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.
Insights
Apurinic/apyrimidinic endonuclease 1 (APE1) regulates G-quadruplex (G4) DNA structures in pancreatic cancer cells. Loss of APE1 reduces KRAS expression and sensitizes cells to chemotherapy, suggesting G4s as therapeutic targets.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Pancreatic ductal adenocarcinoma (PDAC) is aggressive, driven by oncogenic KRAS.
- KRAS expression is regulated by G-quadruplex (G4) DNA structures in its promoter.
- Factors controlling G4 formation and KRAS expression in PDAC remain unclear.
Purpose of the Study:
- Investigate the role of APE1 in regulating KRAS promoter G4 formation.
- Determine if APE1 influences KRAS expression and PDAC cell sensitivity to chemotherapy.
- Assess APE1 and G4 DNA levels in PDAC patient tissues.
Main Methods:
- Biochemical assays to assess APE1 binding to KRAS promoter G4.
- Cellular experiments involving APE1 knockdown to study effects on G4 formation, MAZ transcription factor binding, and KRAS expression.
- In vitro and in vivo drug sensitivity assays.
- Analysis of APE1 and G4 DNA levels in patient samples.
Main Results:
- APE1 is identified as a G4-binding protein that promotes stable G4 formation.
- APE1 binds the KRAS promoter G4 with high affinity, enhancing its folding in vitro.
- APE1 knockdown reduces MAZ binding to the KRAS promoter, decreasing KRAS expression.
- Downregulation of APE1 sensitizes PDAC cells to chemotherapy both in vitro and in vivo.
- Elevated APE1 and G4 DNA levels are observed in PDAC patient tissues.
Conclusions:
- APE1 plays a critical role in establishing stable G4 structures at the KRAS promoter.
- APE1 regulates KRAS expression in PDAC cells.
- G4 structures represent potential prognostic markers and therapeutic targets for PDAC.
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