RADX condenses single-stranded DNA to antagonize RAD51 loading

Abstract

RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) activities is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using singlemolecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA.Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

“…Single-stranded DNA curtains were assembled in microfabricated flowcells according to published protocols (35, 36, 64, 65). All microscope experiments were conducted at 37°C.…”

“…We first generated ssDNA in the flowcells as described previously (35, 65). Next, 0.4 nM RPA-GFP was added to Imaging Buffer (40 mM Tris-HCl pH 8.0, 2 mM MgCl2, 1 mM DTT, 0.2 mg mL -1 BSA, 50 mM NaCl, and 1 mM ATP) and injected at 0.4 mL min -1 to tether the ssDNA molecules at a chromium pedestal 13 μm away from the biotinylated anchors.…”