Premature Cdk1/Cdc5/Mus81 pathway activation induces aberrant replication and deleterious crossover

The error-free DNA damage tolerance (DDT) pathway is crucial for replication completion and genome integrity. Mechanistically, this process is driven by a switch of templates accompanied by sister chromatid junction (SCJ) formation. Here, we asked if DDT intermediate processing is temporarily regulated, and what impact such regulation may have on genome stability. We find that persistent DDT recombination intermediates are largely resolved before anaphase through a G2/M damage checkpoint-independent, but Cdk1/Cdc5-dependent pathway that proceeds via a previously described Mus81-Mms4-activating phosphorylation. The Sgs1-Top3- and Mus81-Mms4-dependent resolution pathways occupy different temporal windows in relation to replication, with the Mus81-Mms4 pathway being restricted to late G2/M. Premature activation of the Cdk1/Cdc5/Mus81 pathway, achieved here with phosphomimetic Mms4 variants as well as in S-phase checkpoint-deficient genetic backgrounds, induces crossover-associated chromosome translocations and precocious processing of damage-bypass SCJ intermediates. Taken together, our results underscore the importance of uncoupling error-free versus erroneous recombination intermediate processing pathways during replication, and establish a new paradigm for the role of the DNA damage response in regulating genome integrity by controlling crossover timing.