Abstract
Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.
Highlights
The human genome is replicated with high fidelity once per cell division cycle
We find that RADX oligomerization is essential for its genome maintenance activities during DNA replication
RADX forms homo-oligomers While purifying MBP-tagged RADX protein expressed in insect cells, we found that concentrated protein elutes from a gel filtration column over a broad range of fractions, with a peak corresponding approximately to the size of a dimer (Figure 1A)
Summary
The human genome is replicated with high fidelity once per cell division cycle. A highly regulated ensemble of proteins involved in DNA synthesis, chromatin deposition, DNA repair, and replication stress responses ensure replication fork stability and completion of genome duplication in a timely manner. RADX is a single-stranded DNA (ssDNA) binding protein (SSB) with similarity to the large subunit of replication protein A (RPA) [16]. RADX inactivation in the absence of any added replication stress causes fork slowing and collapse which can be rescued by inactivating RAD51 [16]. RADX competes with RAD51 for ssDNA, directly binds RAD51, stimulates RAD51 ATPase activity, promotes RAD51 nucleofilament disassembly and inhibits RAD51 recombinase functions [15]. RADX inhibits RAD51-mediated fork reversal, it is important to promote fork reversal in cells exposed to high levels of replication stress [17]. Journal Pre-proof In addition to acting as a regulator of RAD51, the interplay between RADX and RPA on ssDNA is important for replication integrity. We find that RADX oligomerization is essential for its genome maintenance activities during DNA replication
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