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) 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 single-molecule 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.
Highlights
Genomic single-stranded DNA is generated during DNA repair and replication
We adapted the DNA curtain assay for high-throughput single-molecule imaging of RADX-single-stranded DNA (ssDNA) interactions (Figure 1B)
RADX(OB2m) still condenses ssDNA at rates that are indistinguishable from wtRADX, indicating that strong ssDNA binding via the remaining OB-folds is sufficient for naked ssDNA compaction in vitro (Figures S1B, C). These results show that RADX uses its multiple OB-folds to compact ssDNA
Summary
Genomic single-stranded DNA (ssDNA) is generated during DNA repair and replication. During DNA replication, for example, discontinuous synthesis of the lagging strand exposes short stretches of ssDNA that must be protected against nucleolytic degradation. Singlestranded DNA is generated when replication forks stall at DNA lesions or as a result of cellular stress [1, 2]. Fork stability is maintained by diverse ssDNAbinding proteins, including Replication Protein A (RPA), the recombinase RAD51, and RADX. Together, these proteins regulate replication mechanisms to maintain genome stability at stalled replication forks [5, 6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
