The structure-specific endonuclease Mus81–Eme1/Mms4 has an important role in DNA repair and the maintenance of genome integrity (10–12
). However, similar to a ‘double-edged sword’, if the activity of this enzyme was uncontrolled during S-phase, it would lead to the undesired cleavage of some DNA structures during chromosome replication and, consequently, to genomic instability. Therefore, it is essential for cells to possess efficient tools to maintain the nucleolytic function of this endonuclease under strict control. In this work, we have established a regulatory mechanism for Mus81–Eme1/Mms4 that helps to understand how it functions in vivo
. Using budding yeast, we have shown that Mus81–Mms4 is tightly regulated during the mitotic cell cycle by Cdc28 (CDK)- and Cdc5 (Polo-like kinase)-dependent phosphorylation of the Mms4 subunit, and that this phosphorylation is required for the nuclease activity of the complex.
We have found that, whereas Mus81, the catalytic subunit of budding yeast Mus81–Mms4, does not present significant changes throughout the mitotic cell cycle, the non-catalytic subunit, Mms4, undergoes cell cycle-dependent phosphorylation. Mms4 had been previously identified as one of the multiple possible targets of Cdc28 (52
). Here, using a tight, conditional cdc28-td
degron mutant, we have shown that the cell cycle-regulated modification of Mms4 we observed depends on Cdc28. Moreover, our results have also indicated that Mms4 lacking the CDK phosphorylation consensus sites cannot be phosphorylated. These data are consistent with Mms4 being a direct substrate of the cyclin-dependent kinase Cdc28. In addition, Mms4 contains potential conditional docking sites for Polo-like kinases (58
), which overlap with three of the CDK phosphorylation consensus sites of this protein. Our results using a conditional cdc5
mutant have indicated that the Polo-like kinase Cdc5 is also necessary for the phosphorylation of Mms4 during the cell cycle. The involvement of Cdc5 in the modification of this protein is not merely an indirect consequence of the requirement of this kinase for the progression through mitosis, as we have shown that Mms4 is already phosphorylated in cells arrested in G2/M. Taken together, our data strongly suggest that Cdc28 phosphorylates the Mms4 subunit of the Mus81–Mms4 complex, priming this protein for the subsequent phosphorylation by Cdc5. Additionally, it is also possible that Cdc28 phosphorylates and activates Cdc5 (52
), thereby enabling the phosphorylation of Mms4 by this kinase.
The nuclease activity assays carried out in this work have clearly shown that the cell cycle-dependent phosphorylation of Mms4 is required for the full activity of the Mus81–Mms4 complex. The results obtained indicate that Mms4 phosphorylation enables this endonuclease to cleave a variety of branched DNA structures that can be generated under different situations during chromosome replication, including replication forks. Nevertheless, our data have revealed that Mms4 phosphorylation and the subsequent Mus81–Mms4 activation only occur during a narrow window of the cell cycle, once the cells have finished bulk DNA synthesis but before they progress through mitosis. This mode of regulation prevents the nuclease activity of Mus81–Mms4 during S-phase, thus avoiding the potential problems for chromosome replication and genome stability derived from the cleavage of DNA substrates. Moreover, the regulation of this endonuclease provides an efficient safeguard mechanism to resolve, before mitosis, different DNA intermediates that cannot be processed or may escape resolution by other pathways and remain at the end of S-phase. This mechanism contributes to ensure the correct completion of chromosome duplication and the later chromosome segregation, both of which are essential for genome integrity and normal progression through the cell cycle.
The data obtained in the nuclease activity assays with the phosphorylation-defective mms4-np
mutant have provided evidence of the requirement of Mms4 phosphorylation for the normal function of Mus81–Mms4. This mutant has also provided a system to analyse the consequences of defective Mus81–Mms4 activity in cells lacking the RecQ-helicase Sgs1, as the sgs1Δmms4-np
double mutant is viable, whereas the mus81
null mutants are synthetically lethal with sgs1
). The nuclease assays have shown that the mms4-np
cells exhibit reduced endonuclease activity, but this residual function allows cell survival in an sgs1
Δ background. Moreover, unlike mms4
Δ cells, the mms4-np
mutant did not show a significant sensitivity to MMS, HU, 4NQO or cisplatin in SGS1+
cells. This result suggests that the reduced nuclease activity of the mms4-np
cells is sufficient for the cellular response to these agents when Sgs1 is present. However, cells lacking Sgs1 significantly increase their sensitivity to MMS, HU, 4NQO and cisplatin when Mms4 cannot be phosphorylated, indicating that, in the absence of the RecQ helicase, the low nuclease function provided by Mms4-np is not sufficient to cope with the problems originated by these drugs, which results in a hypersensitivity to them. These data indicate that a lack of RecQ-helicase activity makes the function of Mus81–Mms4 crucial, and show that Mms4 phosphorylation is required to confer full activity to the Mus81–Mms4 complex in vivo
. These results also suggest that the non-nucleolytic resolution pathway mediated by the Sgs1/Top3/Rmi1 complex would be the primary choice for cells to resolve intermediates that originate during replication-associated DNA repair or fork stalling, and that Mus81–Mms4, the mode of regulation of which restricts its period of action, would operate later to cleave the unresolved DNA structures.
Our results agree with recent findings on the regulation of Mus81–Mms4 (46
) and expand these studies, providing new insights into the control of Mus81–Mms4 activity during the mitotic cell cycle and its relevance for genome integrity, both under normal conditions and in response to exogenous agents that perturb DNA replication. The data presented here indicate that precise Mus81–Mms4 regulation through the cell cycle plays an essential role in preventing genomic instability, a hallmark of cancer (66
). As Mus81–Mms4 is evolutionarily conserved, it would be interesting to study whether some tumour cells show uncontrolled Mus81 nuclease activity.