Fluorescent human RPA to track assembly dynamics on DNA.

RPA is an essential protein for most DNA metabolic processes including replication, repair, and recombination.RPA is a ssDNA binding protein made of six domains situated across RPA70, RPA32 and RPA14 subunits. Four high affinity DNA binding domains engage the DNA.Site-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics.Bulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA.