Fluorescent human RPA to track assembly dynamics on DNA

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Single-stranded DNA (ssDNA) is central to DNA replication, repair, and recombination.
• Replication Protein A (RPA) is a high-affinity heterotrimeric protein that binds and protects ssDNA.
• RPA acts as a gatekeeper, influencing downstream DNA metabolic pathways and maintaining genomic integrity.

Purpose of the Study:

• To develop methods for studying the dynamic configurations of RPA on ssDNA.
• To enable high-resolution structure-function analysis of RPA-ssDNA interactions.

Main Methods:

• Site-specific incorporation of the non-canonical amino acid 4-azido-L-phenylalanine (4AZP) into human RPA.
• Utilizing click chemistry to generate active site-specific fluorescent RPA.
• Employing Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays.

Main Results:

• Successfully generated site-specific fluorescent human RPA variants.
• Demonstrated the utility of FEncAA and FRET assays for measuring RPA dynamics on DNA.
• Established a versatile method applicable to other multi-domain proteins.

Conclusions:

• Fluorescent RPA provides a powerful tool for dissecting RPA-ssDNA complex dynamics.
• This approach advances the understanding of RPA's role in DNA maintenance and genomic stability.
• The methodology offers broad applicability for studying protein dynamics in molecular biology.