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Updated: Jul 2, 2025
Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair
Published on: May 24, 2017
Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide
Sahiti Kuppa1, Elliot Corless1, Colleen C Caldwell2
1Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Abstract:
Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.
Insights
Researchers developed a fluorescent Xeroderma Pigmentosum group A (XPA) protein to study DNA repair. This new tool helps visualize XPA
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Nucleotide excision repair (NER) maintains genomic stability by removing DNA damage.
- Defects in NER are linked to diseases like Xeroderma Pigmentosum.
- Xeroderma Pigmentosum group A (XPA) protein binding to DNA junctions is crucial for NER.
Purpose of the Study:
- To develop a fluorescent XPA protein for studying its DNA interaction dynamics during NER.
- To establish methods for generating and maintaining this fluorescent probe for biochemical and biophysical assays.
Main Methods:
- Incorporation of 4-azido-L-phenylalanine (4AZP) at Arg-158 in human XPA.
- Conjugation of Cy3 fluorophore to XPA via strain-promoted azide-alkyne cycloaddition, creating XPACy3.
- Validation of XPACy3 DNA binding activity and fluorescence changes.
Main Results:
- Successfully generated a functional fluorescent XPA protein (XPACy3).
- XPACy3 retained its DNA binding capabilities.
- XPACy3 exhibited a significant fluorescence change upon DNA binding, enabling detection.
Conclusions:
- The developed XPACy3 is a valuable tool for investigating NER mechanisms.
- This fluorescent probe facilitates real-time monitoring of XPA-DNA interactions in vitro.
- Established protocols ensure protein stability for detailed biochemical and biophysical studies.
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