Single Molecule Studies of RPA's Sequential Binding to ssDNA Reveals a Highly Stiff and Stable State Induced by the Binding of Zinc

Abstract

Replication protein A (RPA) is a prototypical modular eukaryotic single-stranded DNA (ssDNA) binding protein that has essential biological function in virtually all DNA transactions in cell. In this work we used magnetic tweezers to study the sequential engagement of RPA to single large ssDNA molecule in real time. Using mechanical manipulation of single ssDNA tethers, we found that binding of RPA causes ssDNA to rapidly extend and elongate reaching a steady state within several minutes. Analysis of RPA's effect on the force-extension curve of ssDNA reveals a increased effective persistence length at RPA binding, which indicates a moderate stiffening effect of RPA on ssDNA backbone. Further analysis of RPA's occupation fraction on ssDNA at varying RPA concentration shows that RPA's ssDNA binding is largely non-cooperative with an interesting non-monotonic salt concentration dependent binding affinity. Upon examination of the effect of biometals, we discovered a unique role of zinc on RPA's ssDNA binding: zinc in micromolar concentrations drives formation of a significantly stiffer and more compact RPA coated ssDNA state. These results together provide new mechanochemical insights to the influences and the mechanisms of action of RPA on large single ssDNA substrate.