G2 DNA Synthesis is Essential to Safeguard Chromosome Integrity Under Replication Stress

Incomplete DNA replication caused by replication stress (RS) has been implicated in chromosome fragility, chromosome mis-segregation and genome instability. To counteract this, an unscheduled DNA synthesis pathway, MiDAS, has been proposed to rescue replication stalling after mitotic entry. It remains unclear why stressed replication forks fail to proceed during late interphase. Here we find that, contrary to the MiDAS model, human cells in fact actively maintain DNA synthesis throughout the entirety of G2 phase in face of mild RS. Besides, the persistent DNA replication activity in G2 is necessary for cells to proceed to mitosis. This is because the passage of unresolved replication structures, either in a retarded or stalled state, from S to G2 activates an ATR-dependent replication-stress checkpoint, blocking mitotic entry. Crucially, G2 DNA synthesis alongside associated G2/M delay creates a feedback mechanism to minimise genome under-replication. Acute inactivation of the RS checkpoint in G2 phase causes severe mitotic chromosome malformation; whereas prolonging G2 DNA synthesis effectively suppresses RS-induced DNA damage in offspring cells. Therefore, our study reveals another important role of G2 phase as an “emergency replication window” and, most importantly, demonstrates the biological necessity of DNA replication outside of S phase. This also offer an explanation for why ATR becomes so vital in G2 uniquely in cells experiencing RS. Understanding the plasticity of the replication programme may provide new insights into how (pre)cancerous cells withstand high intrinsic RS.