Single-molecule imaging brings Rad51 nucleoprotein filaments into focus

Abstract

The Rad51 protein is essential for DNA repair by homologous recombination. After DNA damage, Rad51 localizes to nuclear foci that represent sites of DNA repair in vivo. In vitro, Rad51 self-assembles on single- or double-stranded DNA to form a nucleoprotein filament. Recently, the merging of innovative single-molecule techniques with ensemble methods has provided unique insights into the dynamic nature of this filament and its cellular function. The assembly and disassembly of Rad51 nucleoprotein filaments is seen to be regulated by recombination accessory proteins. In this regard, the BRC repeats of the BRCA2 protein were shown to modulate the DNA binding selectivity of Rad51. Furthermore, single-molecule studies explained the need for a DNA translocase, Rad54 protein, in the disassembly of Rad51 double-stranded DNA filaments.

Figure 1. Techniques used to capture and study single molecules of DNA

a) Single optical trap (red) captures a bead (grey) that is attached to a single DNA molecule (green). Force generated by the flow of buffer within a flowcell extends the DNA molecule. b) Two optical traps capture beads on each end of a DNA molecule. c) Magnetic tweezer extends DNA attached to the surface of coverglass through antibody-antigen or biotin-streptavidin interactions. The end of DNA distal to the surface anchor is attached to a magnetic bead. d) DNA molecules tethered to the surface of a coverglass within a flowcell are extended by flow of buffer through the chamber. Modifications on both ends of the DNA result in dual attachment to the surface and will allow the DNA to remain extended in the absence of buffer flow. Alternatively, if DNA is only tethered on the surface at one end, the continuous flow of buffer is required to maintain extension of the DNA for observation. To the right of each illustration (a-d) are actual images obtained with each of the respective techniques.

Figure 2. Illustration showing protein-mediated regulation of Rad51 nucleoprotein filament assembly and disassembly

The BRC repeats of BRCA2 (green ovals) promote binding of Rad51 (blue circles) to ssDNA and inhibit binding of Rad51 to dsDNA. The Rad51 filament formed on ssDNA is stabilized by Rad54 (yellow oval). Rad54 also remodels nucleosomal DNA to permit pairing with the homologous target dsDNA. Following homologous pairing and DNA strand exchange, Rad51 is bound to heteroduplex dsDNA (red and blue DNA molecule). Then, Rad54 promotes heteroduplex extension (branch migration) as well as disassembly of the Rad51-dsDNA filament using its ATP-dependent translocation activity on. The Rad54-mediated disassembly of the Rad51-dsDNA filament allows DNA polymerases and other downstream proteins to access the heteroduplex DNA. Subsequent steps (Figure I Box 1) complete the process.

Figure I. Model for Recombinational Repair of a DNA Double-Strand Break

The Initiation phase involves the resection of the 5′ terminated strand at a DNA double-strand break (red dsDNA molecule). This results in a 3′ terminated ssDNA tail on which the Rad51 nucleoprotein filament is formed. In the next phase, Homologous Pairing and Strand Exchange, the Rad51 nucleoprotein filament locates homology, pairs with the homologous dsDNA template (blue), and exchanges DNA strands. Next, synthesis of new DNA (light blue) using the intact homologous DNA (blue) as a template permits formation of two Holliday junctions. DNA Heteroduplex Extension and Branch Migration involves movement of the Holliday junctions. The final step, Resolution or Dissolution, is the separation of crossover structures either by multiple alternate cleavage events or by convergent migration and associated DNA strand passage of two Holliday junctions to yield two intact repaired homologous DNA molecules.