Impediments that slow the rate of DNA replication are collectively referred to as “replication stress”. Replication stress is the main driver of genome instability in oncogenesis and is recognized as a hallmark of cancer. To cope with replication stress and maintain genomic health, eukaryotic cells have evolved a sophisticated replication stress response pathway that mediates the repair and restart of stalled replication forks.
Actin is a cytoskeletal protein that polymerizes into filaments to provide cells with mechanical support, driving force for shape change and movement, and a substrate for cargo transport. While actin has traditionally been considered a cytoplasmic protein, recent evidence indicates that actin polymerization can also occur within the nucleus. However, the role for nuclear filamentous actin (F-actin), the mechanism(s) triggering its nucleation, and the impact of nuclear actin on the genome remain unclear.
Using a combination of live-cell and super-resolution microscopy, chromatin fibre analysis, biochemistry, and cell and molecular biology, we have discovered that rapid-onset actin polymerization within the nucleus, a process we have termed ‘nuclear F-actin nucleation’ (NFAN), plays a prominent role in the replication stress response. We found that pharmacological inhibition of NFAN resulted in S-phase elongation, reduced DNA replication rate, shortened distance between replication origins, and increased occurrence of micronuclei and anaphase abnormalities, consistent with replication stress. Conversely, pharmacological induction of replication stress induced ataxia telangiectasia and Rad3-related (ATR) and mechanistic target of rapamycin (mTOR)-dependent NFAN, which in turn resulted in the expansion of nuclear volume and redistribution of replication forks to the nuclear periphery. Replication foci in late-S phase that were decorated with the replication stress repair factor Fanconi anaemia complementation group D2 (FANCD2), were found to co-localize with nuclear F-actin. Actin-associated replication forks in late S-phase also migrated along actin filaments with migration rates regulated by ATR and mTOR. Inhibiting NFAN suppressed replication stress-dependent alteration of nuclear structure and prevented the restart of stalled replication forks. Preliminary data indicate that co-localization of stalled replication forks with certain repair factors is spatiotemporally compartmentalized, suggesting that NFAN facilitates movement of stalled forks to the nuclear periphery for repair. Collectively, these data reveal a novel pathway where actin-dependent forces shape the nucleus in response to replication stress to maintain genome health.