A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization - PubMed (original) (raw)

A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization

Xiaolan Zhao et al. Proc Natl Acad Sci U S A. 2005.

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Abstract

Through a genetic screen using myosin-like protein strains mlp1Delta mlp2Delta and biochemical purification, we identified a complex of eight proteins, each required for growth and DNA repair in Saccharomyces cerevisiae. Among the subunits are Mms21 that contains a putative Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain characteristic of small ubiquitin-like modifier (SUMO) ligases, two structural-maintenance-of-chromosome (Smc) proteins, Smc5 and Smc6, and a protein that contains an ubiquitin ligase signature domain. We show that these proteins colocalized to several distinct nuclear foci. Biochemical and genetic data demonstrated that Mms21 indeed functions as a SUMO ligase and that this activity requires the Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain. The substrates for this SUMO ligase include a subunit of the octameric complex, Smc5, and the DNA repair protein Yku70. We further show that the abolition of the SUMO E3 activity of Mms21 leads to such disparate phenotypes as DNA damage sensitivity, defects in nucleolar integrity and telomere clustering, silencing, and length regulation. We propose that Mms21 sumoylates proteins involved in these diverse processes and that the other members of the complex, particularly Smc5/6, facilitate proper substrate sumoylation by localizing Mms21 to specific chromosomal regions.

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Figures

Fig. 1.

Fig. 1.

The deletion of the SP-RING domain of Mms21 is synthetic-lethal with _mlp1_Δ _mlp2_Δ.(A) A diploid strain heterozygous for _mms21_-11, _mlp1_Δ, and _mlp2_Δ was sporulated and dissected. Three tetrads are shown. _mlp1_Δ _mlp2_Δ and _mms21_-11 spore clones are indicated. The genotype of inviable spores was deduced from those of sister spore clones and is _mms21_-_11 mlp1_Δ _mlp2_Δ.(B) Alignment of the SP-RING domains of Siz1, mammalian PIAS enzymes, and Mms21. The eight signature Cys (C) and His (H) residues are indicated above the alignment. Cysteines marked by * vary among different proteins or change to other amino acids. Identical amino acids in the alignment are shaded in gray, and conserved amino acids are underlined. The arrow indicates a transposon insertion in _mms21_-11.

Fig. 2.

Fig. 2.

The Mms21-Smc5-Smc6 complex is composed of eight subunits and forms perinuclear centers. (A) Mms21-ProA was affinity-purified from yeast at the indicated salt concentrations. Copurifying proteins were resolved by SDS/PAGE and stained with Coomassie blue. Individual bands were excised and identified by MS. The identity of each protein is indicated. (B) Subunits of the Mms21-Smc5-Smc6 complex form nuclear foci. Each subunit shown was tagged by YFP at its chromosomal locus. Examples of live-cell fluorescence images are shown for the proteins indicated. Arrowheads mark foci formed by each protein. (C and D) Mms21 foci colocalize with those formed by other subunits of the complex (C), and they are juxtaposed to the nuclear envelope delineated by Nic96 (D). In C, cells contain Mms21-CFP and one of the YFP-tagged subunits as indicated. In D, cells contain Nic96-CFP and Mms21-YFP. Representative live-cell fluorescence images of CFP and YFP fusion proteins as well as their merged pictures are shown. CFP fusion proteins are pseudocolored as red and YFP fusion proteins are green. Arrowheads indicate Mms21-YFP foci in D.

Fig. 3.

Fig. 3.

Defects of the subunits of the Mms21-Smc5-Smc6 complex lead to similar phenotype. (A) Strains containing _mms21_-11 (Upper) or hemagglutinin-tagged NSE4 (Lower) exhibit sensitivity to different DNA damaging agents. Mid-log phase, yeast extract/peptone/dextrose (YPD)-grown cells were spotted in 10-fold serial dilutions (from 105 to 10 cells) on YPD plates or plates containing the indicated amount of DNA-damaging agents. One spotted YPD plate was treated with UV light. All plates were incubated at 30°C for 3 days. (B) A hypomorphic allele of Nse1 exhibits synthetic growth defect when combined with _mlp1_Δ _mlp2_Δ. _mlp1_Δ _mlp2_Δ, NSE1-YFP, and _NSE1-YFP mlp1_Δ _mlp2_Δ strains were streaked out on a YPD plate and grown at 30°C for 3 days. The _mlp1_Δ _mlp2_Δ NSE1-YFP strain grew significantly slower than either the _mlp1_Δ _mlp2_Δ or Nse1-YFP strains.

Fig. 4.

Fig. 4.

Mms21 functions in sumoylation processes. (A and B) _mms21_-11 is synthetic-lethal with ulp1N_Δ_338 and synthetic-sick with _siz2_Δ. Diploid strains heterozygous for _mms21_-11 and ulp1N_Δ_338 (A) or _mms21_-11 and _siz2_Δ (B) were sporulated and dissected. Three tetrads are shown for each case. In A, _mms21_-11 and ulp1N_Δ_338 spore clones are indicated. The genotype of inviable spores was deduced from those of sister spore clones and is _mms21_-11 ulp1N_Δ_338. In B, _mms21_-11 and _mms21_-_11 siz2_Δ spore clones are indicated. _siz2_Δ and WT spore clones grew equally well. (C) _mms21_-11 does not affect the level of the mutated protein. Mms21 (WT) or _mms21_-11 was tagged with Myc tags at their corresponding chromosomal loci. Total yeast lysate was prepared from these cells, separated by SDS/PAGE, and analyzed by immunoblotting using anti-Myc antibody. The nitrocellulose membranes were stained by amidoblack before blotting to check equivalent loading. (D) Yeast SUMO E1 (Aos1/Uba2), E2 (Ubc9), SUMO (Smt3), full-length Mms21, and Mms21 lacking the SP-RING domain (Mms21ΔR) were purified from E. coli via chromatography on Ni-NTA and gel-filtration columns. Protein preparations were subjected to SDS/PAGE analysis to assess purity. (E) Mms21 promotes di-SUMO and tri-SUMO formation in a SP-RING domain-dependent manner. All reactions contain E1 (110 ng), E2 (80 ng), and SUMO (800 ng), and they were carried out at 30°C for 30 min. Additional components included in the reactions are indicated above the gel. Reactions were analyzed by SDS/PAGE and immunoblotting with anti-SUMO antibody. The arrow indicates the position of free SUMO. * and ** depict di-SUMO and tri-SUMO, respectively.

Fig. 5.

Fig. 5.

Mms21 is required for sumoylation of Yku70 and Smc5 in vivo and in vitro.(A) Mms21 is required for sumoylation of Yku70 and Smc5 in vivo.WTor _mms21_-11 cells containing chromosomal Myc-tagged Yku70, Smc5, or Pol30 were grown to early log phase and treated with 0.3% MMS for 2 h. Tagged proteins were affinity-purified from the total lysate by using anti-Myc-conjugated agarose. The eluate was subjected to SDS/PAGE and Western blot analysis using anti-SUMO antibody to detect the sumoylated forms of the protein. The membranes were subsequently stripped and reprobed with anti-Myc antibody to detect the unmodified forms. (B) The Mms21-Smc5 complex, Smc5, and Pol30 were purified from E. coli by chromatography on Ni-NTA and gel-filtration columns. The Yku70-Yku80 complex was purified from yeast by chromatography on a Ni-NTA and a Mono-Q column. Protein preparations were subjected to SDS/PAGE analysis to assess purity. (_C_-E) Mms21 stimulates sumoylation of Yku70 and Smc5 in vitro. All in vitro sumoylation reactions contain E1, E2, and SUMO and were carried out at 30°C for 30 min. Additional components included in the reactions are indicated above the gel. The reactions were analyzed by SDS/PAGE and immunoblotting using antibodies specific to the target proteins. In C, all reactions contain the Yku70-Yku80 complex, and a monoclonal anti-Yku70 antibody was used in the immunoblotting analysis. Arrows indicate the positions of mono- and di-SUMO-Yku70. In D, Smc5 was detected in the immunoblotting analysis by using anti-T7 antibody as Smc5 was fused to the T7 tag. Arrows indicate the positions of mono- and di-SUMO-Smc5. In E, an anti-Pol30 antibody was used in the immunoblotting analysis. Only the unmodified form of Pol30 was detected.

Fig. 6.

Fig. 6.

_mms21_-11 affects nucleolus structure and telomere clustering. (A and B) WT and _mms21_-11 cells containing Nop1-CFP (a nucleolar marker) (A) or Rap1-YFP (a telomere marker) (B) were grown to early log phase. Fluorescence images of live cells were taken at 10 Z-sections (step size, 0.4 μm). Maximum projections of all Z-sections, differential interference contrast images, and the merge of the two are shown. (Insets) Enlarged images of single nuclei are shown. (C) The number of Rap1-YFP foci of 72 WT and 85 _mms21_-11 cells were quantified based on the projections. (D) Telomere position effect increases in _mms21_-11 strains. WT and _mms21_-11 cells containing reporters for telomere silencing (VR TEL:URA3) (44) were spotted in 10-fold serial dilutions on plates that have 5-fluoroorotic acid (5-FOA) (counter selective for Ura3) or lack 5-FOA [synthetic complete (SC) medium, for control]. Partial repression of URA3 expression in WT strains allows some growth on 5-FOA. Complete abolition of silencing in the _sir2_Δ strain results in the failure to grow on 5-FOA (44). Increased silencing in the _mms21_-11 strain has the opposite effect to that of _sir2_Δ.

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