Research Article: Concentration-Dependent Exchange of Replication Protein A on Single-Stranded DNA Revealed by Single-Molecule Imaging

Date Published: February 3, 2014

Publisher: Public Library of Science

Author(s): Bryan Gibb, Ling F. Ye, Stephanie C. Gergoudis, YoungHo Kwon, Hengyao Niu, Patrick Sung, Eric C. Greene, Maria Spies.

http://doi.org/10.1371/journal.pone.0087922

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.

Partial Text

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 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]. Rfa1 contains dbdA, dbdB, and dbdC, which are connected to one another by flexible linkers, and dbdD is found in Rfa2. RPA binds ssDNA in at least three distinct modes: a low affinity mode (Kd∼100 nM) with a binding site size of ∼8 nucleotides, a moderate affinity mode (Kd∼5 nM) with a binding site size of ∼12–23 nucleotides, and a high-affinity mode (Kd∼0.05 nM) with a binding site size of ∼30 nucleotides [3], [4], [5]. In addition, S. cerevisiae RPA exhibits a salt-dependent transition from a binding site of ∼18–20 nucleotides to ∼26–28 nucleotides [9]. 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].

The ability to directly visualize the assembly of individual nucleoprotein complexes in real time offers a powerful approach for dissection of complex multi-component reactions pathways such as homologous DNA recombination. Here we have used total internal reflection fluorescence microscopy to visualize single-stranded DNA curtains bound by either RPA-eGFP or RPA-mCherry, and we use the displacement of these fluorescent versions of RPA as a read-out for the dynamic properties of RPA as well as the assembly of wild-type Rad51 presynaptic filaments. This system allows for temporally controlled delivery of reaction components, and recapitulates several known attributes of presynaptic filament assembly along with new, unanticipated behaviors of RPA.

 

Source:

http://doi.org/10.1371/journal.pone.0087922