Interplay between the Smc5/6 complex and the Mph1 helicase in recombinational repair

Abstract

The evolutionarily conserved Smc5/6 complex is implicated in recombinational repair, but its function in this process has been elusive. Here we report that the budding yeast Smc5/6 complex directly binds to the DNA helicase Mph1. Mph1 and its helicase activity define a replication-associated recombination subpathway. We show that this pathway is toxic when the Smc5/6 complex is defective, because mph1Delta and its helicase mutations suppress multiple defects in mutants of the Smc5/6 complex, including their sensitivity to replication-blocking agents, growth defects, and inefficient chromatid separation, whereas MPH1 overexpression exacerbates some of these defects. We further demonstrate that Mph1 and its helicase activity are largely responsible for the accumulation of potentially deleterious recombination intermediates in mutants of the Smc5/6 complex. We also present evidence that mph1Delta does not alleviate sensitivity to DNA damage or the accumulation of recombination intermediates in cells lacking Sgs1, which is thought to function together with the Smc5/6 complex. Thus, our results reveal a function of the Smc5/6 complex in the Mph1-dependent recombinational subpathway that is distinct from Sgs1. We suggest that the Smc5/6 complex can counteract/modulate a pro-recombinogenic function of Mph1 or facilitate the resolution of recombination structures generated by Mph1.

Fig. 1.

Mph1 interacts with the Smc5/6 complex but is not sumoylated. (A) Mph1 interacts with Smc5 in 2H. The 2H strain pJ69–4a containing either a GAD-Mph1 or a GAD vector was mated to pJ69–4alpha cells containing GBD-fused Nse1–6 (lanes 1–6), Smc5 (lane 7), and Smc6 (lane 8). The resulting diploids were selected on -TRP-LEU (-T-L) plates, and reporter activation was scored by replica plating to -TRP-LEU-HIS (-T-L-H) and -TRP-LEU-ADE (-T-L-A) media. Note that GBD-Nse2/Mms21 self-activates the HIS3 and ADE2 reporters. White boxes indicate the interactions between Mph1 and Smc5 and the corresponding controls. (B–D) Mph1 co-immunoprecipitates with Smc5, Smc6, and Nse6. Lysates from cells containing the indicated tagged constructs were immunoprecipitated with the indicated antibody. Cell lysates (input) and immunoprecipitated proteins (IP) were analyzed by protein blotting using anti-Flag (Upper) and anti-Myc (Lower) antibodies. In (B), Smc5 was precipitated by anti-Flag antibody only in the presence of Mph1-Flag, and vice versa. In (C and D), Mph1 was precipitated by anti-Myc antibody only in the presence of either Smc6-Myc (C) or Nse6-Myc (D). (E) Mph1 binds Smc5 in vitro. Recombinant Mph1-GST and Smc5-His₆ were expressed in E. coli, and Mph1-GST was pulled down by the Ni-NTA resins only in the presence of Smc5-His₆. E: eluate, W: wash. (Top) Coomassie stained gel; (Bottom) Western blot using anti-GST antibody. (F) Smc5, but not Mph1, is sumoylated. Cells containing Myc-tagged Mph1 or Smc5 were grown in the absence (−MMS) or presence (+MMS) of MMS. Mph1 and Smc5 were immunopurified by anti-Myc antibody and examined by protein blotting using anti-Myc (Bottom) and anti-SUMO antibodies (Top).

Fig. 2.

mph1Δ rescues several defects of smc6 and mms21 mutants, whereas Mph1 overexpression confers opposite effects. (A and B) mph1Δ rescues the growth defects and DNA damage sensitivity of smc6–56 (A), smc6-P4, and mms21–11 (B). WT and mutant cell cultures were diluted and spotted onto plates containing no drug or the indicated concentration of drugs or were treated with the indicated dose of UV light. (C and D) mph1Δ rescues centromere separation defects in smc6–56 cells. Cells were arrested in G1 at 23 °C and were shifted to 37 °C for 1 h to inactivate the smc6–56 allele before being released from G1. Samples were collected every 15 min to examine cell-cycle progression by FACS and chromatid separation by the appearance of 2 dots of GFP-LacI bound to tandem LacO repeats near the centromere on chromosome IV. To examine the events in the first cell cycle only, alpha-factor was added back to the cultures 45 min after release. (E and G) Mph1 overexpression sensitizes cells with defective Smc5/6 complexes. Cells containing smc6-P4 or mms21–11 (E) or SMC6-YFP (SMC6-Y, G) were transformed with pGAL-Mph1 or the control vector; cell cultures were diluted and spotted on plates lacking uracil with glucose or galactose and with and without MMS or HU. (F) Smc6-YFP confers normal growth but is synthetic sick with mms21–11. A representative tetrad from diploid strains heterozygous for SMC6-YFP and mms21–11 is shown. The genotype for each spore clone is indicated. (H) mph1Δ suppresses the lethality of mms21Δ and smc6Δ cells. Representative tetrads from diploid strains with the indicated genotypes are shown. The spore clones containing mms21Δ mph1Δ (Left) and smc6Δ mph1Δ (Right) are underlined; those containing mms21Δ (Left) and smc6Δ (Right) are marked with dotted lines, and their genotypes are deduced from sibling spore clones.

Fig. 3.

A pro-recombinogenic function of Mph1 is toxic in mutants of the Smc5/6 complex. (A) Mph1 foci co-localize with those of Rad52 and Pol30. Representative images of WT cells containing Mph1-YFP, Rad52-RFP, and CFP-Pol30 are shown. Foci formed by each protein are indicated by arrows. (B) rad51Δ, like mph1Δ, suppresses the sensitivity to MMS and HU of smc6-P4 cells (Left) and smc6–56 cells (Right). Experiments were performed as in Fig. 2A. (C and D) Helicase mutants mph1-E210Q and -Q603D exhibit sensitivity to MMS similar to that of mph1Δ (C) and suppress the sensitivity of smc6–56 cells to MMS and HU (D). Experiments were carried out as described in Fig. 2A. (E) Mph1-E210Q and -Q603D protein levels are similar to that of WT Mph1. WT and mutant Mph1 proteins were tagged with YFP at their chromosomal loci; their protein levels were examined by protein blotting using anti-YFP antibody (Top). Similar loading was verified by amido-black staining (Bottom). (F) Similar amounts of WT Mph1 and Mph1-Q603D are immunoprecipitated with Smc5. Experiments were carried out as in Fig. 1B.

Fig. 4.

mph1Δ and mph1-Q603D decrease the levels of recombination intermediates in smc6 and mms21 mutants. (A and B) Cells were arrested at G2/M phase with nocodazole at 25 °C and then were released into YPD medium with 0.033% MMS at 30 °C. The replication and recombination intermediates at the ARS305 region 60, 120, and 180 min after release were analyzed by 2D gel electrophoresis followed by Southern blotting. Diagrams indicating the position of the probe and the replication structures are shown above the 2D gel images. The X-shaped DNA structures are indicated by arrowheads in smc6 and mms21 mutants. FACS analyses are presented to the right of each gel image.

Fig. 5.

Genetic interactions of mph1Δ and mms21–11 with sgs1Δ and srs2Δ and models for the functions of the Smc5/6 complex and Mph1 in recombinational repair. (A) The amount of X-shaped DNA in sgs1Δ cells is largely unaffected by mph1Δ. Experiments were carried out as in Fig. 4. (B and D) mph1Δ enhances the MMS sensitivity of sgs1Δ (B) and srs2Δ (D) cells, and srs2Δ exacerbates MMS sensitivity of mms21–11 cells (D). Experiments were carried out as in Fig. 2A, except that plates were incubated for three days in B. (C) The synthetic sick interaction between sgs1Δ and mms21–11 is alleviated by rad51Δ. Shown are 3 tetrads from indicated diploids; the spore clones of the relevant genotype are indicated. (E) Models for the functions of the Smc5/6 complex and Mph1 in recombinational repair. See text for details. RFs: replication forks, HR: homologous recombination.