Actin nucleation safeguards single-stranded DNA and promotes replication fork protection

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress and protect stalled replication forks. All such responses rely on the formation of Replication Protein A (RPA)-ssDNA complexes, yet supra-physiological binding of RPA to ssDNA is toxic. How cells regulate RPA availability to promote fork protection and genome stability is largely unknown. Here we establish that during replication excess RPA is sequestered by monomeric actin and released upon replicative stress through transition to polymeric actin state. Impairment in actin nucleation leads to RPA sequestration, deprotection of ssDNA generated at the stressed forks and consequently, catastrophic fork collapse and hypersensitivity to replication inhibitors. In line with this, we show that increasing RPA load is sufficient to restore efficient fork protection in actin polymerization mutants. Collectively, this work identifies a simple yet robust RPA-buffering mechanism regulating its availability to bind ssDNA and protect replication forks against nucleolytic degradation. Inhibition of this pathway could be of therapeutic interest in treatment of cancers.