Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids.

ABSTRACT: An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding

SUBMITTER: Pokhrel N 

PROVIDER: S-EPMC5766198 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature