Faculty Collaboration Database

Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids. Nucleic Acids Res 2017 Sep 19;45(16):9413-9426

Date

09/22/2017

Pubmed ID

28934470

Pubmed Central ID

PMC5766198

DOI

10.1093/nar/gkx598

Scopus ID

2-s2.0-85031928912 (requires institutional sign-in at Scopus site)   26 Citations

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 proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance.

Author List

Pokhrel N, Origanti S, Davenport EP, Gandhi D, Kaniecki K, Mehl RA, Greene EC, Dockendorff C, Antony E

Authors

Edwin Antony PhD Assistant Professor in the Biology department at Marquette University
Christopher Dockendorff PhD Assistant Professor, Organic and Medicinal Chemistry in the Chemistry department at Marquette University

MESH terms used to index this publication - Major topics in bold

Azides
DNA, Single-Stranded
Fluorescent Dyes
Homologous Recombination
Microscopy, Fluorescence
Phenylalanine
Rad51 Recombinase
Replication Protein A
Saccharomyces cerevisiae Proteins
Tryptophan