Real Time Visualization of hRPA Binding to Torsionally Controlled Double-Stranded DNA

Abstract

In eukaryotic cells, single-stranded DNA (ssDNA) is rapidly bound and stabilized by ssDNA-binding proteins (SSBs). This prevents ssDNA from binding back on itself into secondary structures. The main eukaryotic SSB is replication protein A (RPA), which is important for repair, replication and recombination. We study the dynamics of human RPA (hRPA) on topologically constrained DNA at the single-molecule level with magnetic tweezers. This assay allows us to apply varying torsional stress and stretching forces on the dsDNA, parameters that are known to influence the hRPA unwinding reaction. We are interested in uncovering positional preference, cooperativity and directionality of hRPA-DNA binding. To observe this directly, we visualize the position(s) of the hRPA along the DNA, using a combination of magnetic tweezers with fluorescence microscopy. With magnetic tweezers a dsDNA molecule is held between a glass surface and a magnetic bead by a pair of magnets. The stretching force and torsional stress is controlled by the distance and rotations of the magnets. The molecule is pulled sideways and the fluorescently labeled RPA is imaged with an inverted microscope. By applying negative supercoiling at high forces, bubbles of ssDNA open up. Multiple bubbles will allow RPA to bind to multiple sites, and such RPA-stabilized bubbles may coexist or slowly anneal into one larger bubble. Our first experiments show that more than one RPA-binding sites occurs when assembled on 20 kb dsDNA that is underwound. These RPA-bound spots appear to be stable in time. We will report the structure and dynamics of RPA-stabilized ssDNA bubbles at the meeting.