Abstract
Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance [1], [2], [3]. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA.
Highlights
Replication protein A (RPA) is a heterotrimeric complex consisting of Rfa1 (70 kDa), Rfa2 (32 kDa), and Rfa3 (14 kDa), and the complex contains a total of six oligonucleotide/oligosaccharide (OB) folds, four of which are involved in single-stranded DNA (ssDNA) binding [3,4,5]
DNA Curtain assay for RPA-eGFP-ssDNA filaments We have established DNA curtains as a method for aligning large numbers of lipid-tethered DNA molecules at the leading edges of nanofabricated chromium (Cr) barriers within a microfluidic sample chamber where they can be visualized by total internal reflection fluorescence microscopy (TIRFM)[36,37,38,39]
RPA-eGFP was injected into the sample chamber, and the downstream ends of the resulting RPA-ssDNA complexes were anchored through nonspecific adsorption to exposed Cr surfaces, allowing the eGFP-tagged complexes to be visualized by total internal reflection fluorescence (TIRF) microscopy in the absence of buffer flow (Figure 1C & Video S1)[40]
Summary
RPA is a heterotrimeric complex consisting of Rfa (70 kDa), Rfa (32 kDa), and Rfa (14 kDa), and the complex contains a total of six oligonucleotide/oligosaccharide (OB) folds, four of which are involved in ssDNA binding [3,4,5]. RPA binds tightly to ssDNA with a defined polarity and the four DNA-binding domains are termed dbdA, dbdB, dbdC, and dbdD [3,4,5,6,7,8]. Rfa contains dbdA, dbdB, and dbdC, which are connected to one another by flexible linkers, and dbdD is found in Rfa. It has been suggested that these different binding modes may reflect the sequential association of distinctly ordered subsets of DNA-binding domains, which may facilitate initial binding to ssDNA as well as the displacement from ssDNA by other ssDNA-binding proteins [4]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
