Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1.

Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome.