DNA replication is highly resilient and persistent under the challenge of mild replication stress

Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild replication stress (RS). Contrary to this proposal, we find that cells exposed to mild RS in fact maintain continued DNA replication throughout G2 and during G2-M transition in RAD51- and RAD52-dependent manners. Persistent DNA synthesis is necessary to resolve replication intermediates accumulated in G2 and disengage an ATR-imposed block to mitotic entry. Because of its continual nature, DNA synthesis at very late replication sites can overlap with chromosome condensation, generating the phenomenon of mitotic DNA synthesis. Unexpectedly, we find that the commonly used CDK1 inhibitor RO3306 interferes with replication to preclude detection of G2 DNA synthesis, leading to the impression of a mitosis-driven response. Our study reveals the importance of persistent DNA replication and checkpoint control to lessen the risk for severe genome under-replication under mild RS.