Related Experiment Video
Updated: Jul 21, 2025
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
A FRET-Based Assay for the Identification of PCNA Inhibitors
Sarah Hardebeck1, Sebastian Schreiber1, Annika Adick2
1University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, Pharmacampus, 48149 Münster, Germany.
Abstract:
Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.
Insights
A new FRET assay analyzes the Proliferating Cell Nuclear Antigen (PCNA)-p15 interaction, crucial for DNA repair. It revealed PCNA stability issues in a rare neurodegenerative disease variant, not altered affinity.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Proliferating cell nuclear antigen (PCNA) is central to DNA metabolism, interacting with proteins like p15 to regulate replication and repair.
- The PCNA-p15 interaction is a potential therapeutic target for cancer treatment.
- Understanding PCNA variants is crucial for diseases linked to DNA repair deficiencies.
Purpose of the Study:
- Develop a Förster resonance energy transfer (FRET)-based assay to analyze the PCNA-p15 interaction.
- Utilize the assay for screening PCNA-p15 interaction inhibitors and studying mutation effects on affinity.
- Investigate the impact of the PCNA C148S variant, associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2), on PCNA-p15 interaction and protein stability.
Main Methods:
- Development of a FRET-based assay for PCNA-p15 interaction analysis.
- Incubation time-dependent FRET measurements.
- Intrinsic tryptophan fluorescence spectroscopy.
- Differential scanning fluorimetry (DSF).
- Asymmetrical flow field-flow fractionation (AF4).
Main Results:
- The developed FRET assay effectively analyzes the PCNA-p15 interaction and its modulation by mutations.
- PCNA C148S variant showed no change in p15 binding affinity.
- PCNA C148S exhibited impaired stability and increased aggregation, confirmed by multiple biophysical techniques.
- The assay's utility was demonstrated in analyzing a disease-associated PCNA variant.
Conclusions:
- A versatile FRET assay for PCNA-p15 interaction analysis was successfully developed.
- The PCNA C148S variant's disease association with ATLD2 is linked to impaired PCNA stability and aggregation, not altered p15 binding.
- The assay is valuable for drug discovery targeting PCNA-p15 and for characterizing disease-related PCNA variants at a molecular level.
