Replication origin under-licensing extends S phase and alters the replication program. Consequently, cells enter mitosis with incompletely-duplicated chromosomes.

Maintaining a stable genome is paramount for cell identity, function and speciation. Conversely, mutations and chromosome rearrangements sustain evolution and adaptation of cells. How cells set the proper balance between genome stability and instability is poorly understood. Loci that naturally replicate late during S phase show increased rates of mutations and rearrangements. By tuning down replication origin licensing in yeast, which extends S phase without perturbing fork progression nor activating replication checkpoint, we explored how late replication triggers chromosome rearrangements. We uncovered a key role for DNA polymerase alpha (Pola) phosphorylation in allowing the completion of DNA synthesis in early mitosis. Mitotic kinases phosphorylate and evict Pola from replisomes, causing ssDNA accumulation that potently activates the replication stress checkpoint. This mechanism provides time for cells to complete DNA synthesis, creating both SNP and SV. This work identifies mitotic Pola phosphorylation as a key switch for remodelling replication forks into recombination intermediates when cells enter mitosis with incompletely replicated chromosomes.