DNA replication is a vulnerable process that must proceed without error to ensure accurate genome copying with each cell cycle. Perturbations in DNA replication, which lead to slowing or collapse of replication forks, is defined as “replication stress”. Replication stress is the main driver of genome instability in early cancer development, and is recognized as one of the hallmarks of cancer. Understanding the molecular basis of replication stress, and the pathways that respond when replication is impaired, is therefore crucial for the understanding of tumorigenesis. In this project, we have uncovered a novel mechanism of nuclear-cytoplasmic cross-talk, regulated by the actin cytoskeletal network, which guards genome stability in response to replication stress.

Actin is a cytoskeletal protein that forms filaments to provide cells with mechanical support and driving forces for movement. While actin was traditionally considered a cytoplasmic protein, recent evidence indicates actin polymerization also occurs inside the nucleus. However, the role for actin fibers in nucleus, and the mechanism that triggers their polymerization, remain unknown.

Using live-cell and super-resolution imaging, chromatin fiber analysis, biochemistry, cell and molecular biology, we have discovered that actin polymerization plays a prominent role in the cellular response to replication stress. We have found that following replication stress, the DNA damage response kinase ATR, regulates the mTOR signaling pathway to drive actin polymerization in both the cytoplasm and nucleus. Directly measuring replication dynamics with sensitive chromatin fiber analysis, revealed that actin polymerization is required for efficient restart of stalled DNA replication. As actin-dependent repair of stalled replication is proceeding, actin polymerization also drives nuclear volume changes, and movement of DNA replication forks to the nuclear periphery. We anticipate stalled replication forks move to the nuclear edge for repair, where replication fork stabilization and restart have been shown to take place. Cumulatively, our data reveal that actin dependent forces shape the nucleus in response to replication stress, and mediate repair of stalled replication, to maintain a healthy genome.

Cytoplasmic-nuclear cross-talk has historically focused on understanding how external cellular inputs are signaled through the cell body, to the nucleus, to drive transcriptional changes. Here we have found that nuclear-cytoplasmic crosstalk signals in the opposite direction to engage multiple cellular compartments in defense of the deleterious consequences caused by replication stress.