Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids.
Nucleic Acids Res
; 45(16): 9413-9426, 2017 Sep 19.
Article
en En
| MEDLINE
| ID: mdl-28934470
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.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Proteínas de Saccharomyces cerevisiae
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Proteína de Replicación A
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Recombinación Homóloga
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos