Abstract
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.
Highlights
Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress
We find that the Dna[2] helicase activity acts on replication fork stalling, promoting full genome duplication along a pathway parallel to homologous recombination (HR)-mediated replication fork recovery
When we determined the effect of checkpoint disruption on cell viability, we found that deletion of RAD9 increased the viability of Dna[2] helicase-defective cells after acute replication stress treatment threefold, reaching levels very similar to those observed for the rad9D control strain
Summary
Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. An analogous reaction, mediated by the DNA2 nuclease in conjunction with Werner’s syndrome helicase WRN, promotes replication restart in human cells[23], while failure to properly control DNA2-mediated DNA resection at stalled forks leads to excessive DNA degradation and genome instability[24,25]. A number of Dna[2] mutants affected within the conserved SF1 helicase motifs I-VI confer growth defects accompanied by sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS)[14,26] This phenotype is not generally shared with mutants affected in the N-terminal domain[26] or nuclease domain[27], indicating that Dna[2] helicase-specific functions in the repair of DNA damage or in the response to damage-induced replication stress exist
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