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. 2012 Sep 1;40(17):8325-35.
doi: 10.1093/nar/gks599. Epub 2012 Jun 22.

Cell Cycle-Dependent Regulation of the Nuclease Activity of Mus81-Eme1/Mms4

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Free PMC article

Cell Cycle-Dependent Regulation of the Nuclease Activity of Mus81-Eme1/Mms4

María Gallo-Fernández et al. Nucleic Acids Res. .
Free PMC article

Abstract

The conserved heterodimeric endonuclease Mus81-Eme1/Mms4 plays an important role in the maintenance of genomic integrity in eukaryotic cells. Here, we show that budding yeast Mus81-Mms4 is strictly regulated during the mitotic cell cycle by Cdc28 (CDK)- and Cdc5 (Polo-like kinase)-dependent phosphorylation of the non-catalytic subunit Mms4. The phosphorylation of this protein occurs only after bulk DNA synthesis and before chromosome segregation, and is absolutely necessary for the function of the Mus81-Mms4 complex. Consistently, a phosphorylation-defective mms4 mutant shows highly reduced nuclease activity and increases the sensitivity of cells lacking the RecQ-helicase Sgs1 to various agents that cause DNA damage or replicative stress. The mode of regulation of Mus81-Mms4 restricts its activity to a short period of the cell cycle, thus preventing its function during chromosome replication and the negative consequences for genome stability derived from its nucleolytic action. Yet, the controlled Mus81-Mms4 activity provides a safeguard mechanism to resolve DNA intermediates that may remain after replication and require processing before mitosis.

Figures

Figure 1.
Figure 1.
Mms4 undergoes cell cycle-dependent phosphorylation. (A) MUS81-HA and HA-MMS4 cells were blocked in G1 using α factor and then released from the block and monitored for approximately two cell cycles. The cells were collected at the indicated time points and the DNA content was determined by flow cytometry. (B) Percentage of budded and binucleated cells throughout the experiment. (C) Immunoblot analysis of the Mus81-HA and HA-Mms4 proteins throughout the experiment. A Pounceau S stained membrane coincident with Mus81/Mms4 migration was used as a loading control. (D) HA-MMS4 cells were blocked in G1 using α factor and then released into S-phase either in the absence or the presence of nocodazole in the medium. The cells were collected at the indicated time points, and the DNA content was determined by flow cytometry. (E) Percentage of budded and binucleated cells at each time point. (F) Immunoblot analysis of HA-Mms4 during the course of the experiment. (G) Phosphatase assay. HA-Mms4 was immunoprecipitated from extracts obtained from nocodazole-arrested cells. The protein was then incubated with or without λ-phosphatase and with λ-phosphatase plus phosphatase inhibitors prior to immunoblot analysis. Phosphorylated Mms4 is indicated as ‘Mms4-P’.
Figure 2.
Figure 2.
Mms4 phosphorylation through the cell cycle is Cdc28 and Cdc5 dependent. (A) Mms4 phosphorylation depends on Cdc28. Scheme of the experiment as explained in the text. (B) cdc28-td PGAL1–10-UBR1 HA-MMS4 cells were collected at the indicated time points, and the DNA content was determined by flow cytometry. (C) Immunoblot analysis of HA-Mms4, Myc-Ubr1 and HA-Cdc28-td during the course of the experiment. The phosphorylated form of Mms4 is indicated as ‘Mms4-P’. The asterisk indicates an unrelated band that cross-reacted with the 12CA5 antibody. A Pounceau S-stained membrane was used as a loading control. (D) Mms4 phosphorylation requires Cdc5 activity. Scheme of the experiment as explained in the text. (E) The cell cycle progression of cdc5-1 HA-MMS4 cells was monitored by flow cytometry. (F) Immunoblot analysis of HA-Mms4 during the course of the experiment.
Figure 3.
Figure 3.
Mms4 phosphorylation correlates with the acquisition of nuclease activity by Mus81–Mms4. The extracts were prepared from MMS4-TAP cells synchronized in G1 with α factor, in S-phase with HU and in G2/M with nocodazole. Mms4-TAP was analysed by immunoblot (A–C, upper panel). In each case, Mms4-TAP was immunoaffinity purified from the extracts, and the nuclease activity (lower panel) was assayed by the resolution of three different 32P-labelled substrates: a 3′-flap (3′-FL) (A), a replication fork (RF) (B) and a nHJ (C). An arrow indicates the labelled-product resulting from the nucleolytic cleavage of each substrate. The controls were nuclease assays using the immunoprecipitated extracts from untagged cells blocked in G2/M or a reaction in the absence of extract.
Figure 4.
Figure 4.
Reduced nuclease activity in a phosphorylation-defective mms4 mutant. (A) HA-mms4-np cells were blocked in G1 using α factor and released from the block in fresh medium. Cells were collected at the indicated time points and the DNA content throughout the cell cycle was monitored using flow cytometry. (B) Percentage of budded and binucleated cells during the experiment. (C) Immunoblot analysis of HA-Mms4-np during the course of the experiment. (D) Nuclease activity assay. The extracts were prepared from HA-mms4-np and HA-MMS4 cells blocked in G2/M with nocodazole. The phosphorylation of wild-type Mms4 and mutant Mms4-np in the whole cell extract (WCE), as well as the yield of the immunoprecipitation of each protein (IP) were monitored by immunoblot (upper panels). About 2% of the total amount of the immunoprecipitated protein used for the nuclease assays was loaded in each case. The nuclease activity was assayed using a 32P-labelled 3′-flap as a substrate (lower panel). An arrow indicates the labelled-product resulting from the nucleolytic cleavage. The controls were as in Figure 3.
Figure 5.
Figure 5.
Cells lacking the RecQ-helicase Sgs1 increase the sensitivity to agents that cause DNA damage or replicative stress in the absence of Mms4 phosphorylation. Serial dilutions (10-fold) of normalized log-phase cultures of the different strains were spotted onto YPD plates containing the indicated amounts of MMS, 4NQO, HU or cisplatin and incubated at 30°C for 48–72 h. The HA-MMS4 and sgs1ΔHA-MMS4 strains, which behave like wild-type and sgs1Δ, respectively, were used as controls to show that the phenotypes of sgs1Δ-HA-mms4-np cells are not due to the tag on Mms4.

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