Abstract
Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA.
Highlights
Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell
To obtain the conformational information in the absence of force, we sought to use another deposition method that has been demonstrated to introduce less perturbation to protein-DNA complexes. This method uses glutaraldehyde-coated mica surface to crosslink the protein-DNA complex formed in solution to the mica surface[39], which has been demonstrated to have less perturbation to DNA-protein complexes compared with other deposition methods such as APTES-mica and freshly cleaved mica[39,40]
These results reveal that RPA binding leads to a more extended ssDNA conformation, which depends on the protein concentration, as quantified by the total extension elongation until the steady state is reached (Fig. 2C)
Summary
Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. We discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA. As protein-DNA interaction typically involves a binding energy of several kBT and nm range interaction distances, forces of a few pN are expected to exist on chromosomal DNA due to DNA packaging
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