Topological stress regulates replication fork dynamics in unperturbed S phase

Accurate genome duplication requires tight-regulation of replication fork progression, and disruptions to this process are a major source of genomic instability, yet how fork dynamics are controlled during unperturbed S-phase remains unclear. We found replication forks elongate slowly in early S (ES) and faster in late S, independent of transcription or nucleotide availability. Elevated origin firing coupled with low TOP2A in ES generates torsional stress, causing replisome uncoupling, reduced fork speed, and basal ATR-CHK1 activation. Overexpression of TOP2A enhances fork speed and reduces replication stress in ES. Thus, TOP2A is a limiting replication factor during unperturbed ES, and basal ATR-CHK1 signaling is driven by transient replisome uncoupling. Also, TOP2A overexpression suppresses oncogene-driven replication stress. Given that TOP2A is frequently upregulated in cancers, it may function as a compensatory response to oncogene-induced replication stress. Together, these findings establish TOP2A as a central regulator of replication fork dynamics. Regulation of replication fork speed is vital for genome integrity. Here, the authors show that limiting TOP2A levels in early S phase induces topological stress and fork slowing whereas TOP2A overexpression accelerates replication while enhancing genome stability.