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. 2006 Feb;172(2):783-94.
doi: 10.1534/genetics.105.047167. Epub 2005 Oct 3.

SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II

Yoshimitsu Takahashi  1 , Vladimir Yong-Gonzalez, Yoshiko Kikuchi, Alexander Strunnikov
Affiliations

SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II

Yoshimitsu Takahashi et al. Genetics. 2006 Feb.

Abstract

The Smt3 (SUMO) protein is conjugated to substrate proteins through a cascade of E1, E2, and E3 enzymes. In budding yeast, the E3 step in sumoylation is largely controlled by Siz1p and Siz2p. Analysis of Siz- cells shows that SUMO E3 is required for minichromosome segregation and thus has a positive role in maintaining the fidelity of mitotic transmission of genetic information. Sumoylation of the carboxy-terminus of Top2p, a known SUMO target, is mediated by Siz1p and Siz2p both in vivo and in vitro. Sumoylation in vitro reveals that Top2p is an extremely potent substrate for Smt3p conjugation and that chromatin-bound Top2p can still be sumoylated, unlike many other SUMO substrates. By combining mutations in the TOP2 sumoylation sites and the SIZ1 and SIZ2 genes we demonstrate that the minichromosome segregation defect and dicentric minichromosome stabilization, both characteristic for Smt3p-E3-deficient cells, are mediated by the lack of Top2p sumoylation in these cells. A role for Smt3p-modification as a signal for Top2p targeting to pericentromeric regions was suggested by an analysis of Top2p-Smt3p fusion. We propose a model for the positive control of the centromeric pool of Top2p, required for high segregation fidelity, by Smt3p modification.

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Figures

Figure 1.
Figure 1.
Minichromosome maintenance phenotype of SUMO E3 mutants. (A) Siz− mutants destabilize mitotic transmission of minichromosomes. YCplac111 stability was determined in the Siz+ (BY4733) and Siz− (4bAS399) strains at 30° as described in materials and methods. (B) Siz− mutants do not destabilize mitotic transmission of acentric plasmids. The stability of pAS255 (replicative), pRS426 (2μ ORI), and pIA1 (Flp− 2μ) plasmids in the Siz+ (BY4733) and Siz− (4bAS399) strains was determined at 30° as described in materials and methods.
Figure 2.
Figure 2.
Top2p is modified in a SUMO E3 dependent manner. (A) Chromatin-bound Top2p is a sumoylation substrate in vitro. All putative targets are HA-tagged. Reaction mixtures with 1 μl of chromatin purified from yeast cells BY4733/pYT1033 (Top2), BY4733bp5 (Pds5), YPH499bp1 (Smc4), YPH499bp2 (Smc2), YPH499bp6 (Brn1), BY4733bp4 (Ycs4), and YPH499bp5 (Ycs5) were incubated at 37° for 120 min (incub. +) and subjected to immunoblotting with anti-HA antibodies. Identical reaction mixtures held on ice (incub. −) were used as negative controls. (B) Purified Top2p is modified by Smt3p in vitro. A total of 4.4 μg purified Top2p (Vaughn et al. 2005) was subjected to sumoylation in vitro (incub. +) or left on ice (incub. −) (see materials and methods) at 37° for 60 min. Western blotting was done with anti-Top2p antibodies. (C) Smt3p-modified Top2p remains strongly associated with chromatin. Top2p was modified in chromatin context in vitro as described in materials and methods. The mock reaction (Smt3 −) was carried out in the absence of recombinant Smt3p. IN, reaction before extraction. Extraction of Top2p after sumoylation reaction was performed for 30 min at 4° with EBX or EBX + 0.5 m NaCl buffers. Chromatin (P) and soluble fractions (S) were separated by centrifugation and analyzed by Western blotting. The Top2p–Smt3p conjugates are marked with an asterisk. (D) Top2p is modified by Smt3p in vitro. A total of 5 μl of chromatin from BY4733/pYT1033 (Top2) or BY4733/pYT1051 (Top2–Smt3) were incubated with the in vitro sumoylation reaction mix (see materials and methods) at 37° for 60 min and subjected to immunoblotting with anti-HA antibodies. The characteristic mobility shift caused by sumoylation corresponds to the shift generated by Smt3p fusion. The Top2p–Smt3p conjugates are marked with an asterisk. (E) The COOH-terminal consensus sumoylation sites of Top2p are the primary targets of Smt3p conjugation in vitro. Reaction mixtures with 5 μl of chromatin purified from yeast cells BY4733/pYT1033 (Top2) and BY4733/pYT1032 (Top2–SNM), containing HA-tagged Top2p, were incubated at 37° for 60 min and subjected to immunoblotting with anti-HA antibodies. (F) Top2p–Smt3p conjugation in vitro is stimulated by SUMO E3. A total of 5 μl of chromatin (sub.) from 4bAS399/pYT1033 (Siz−) was incubated (incub.) with the in vitro sumoylation reaction mix (see materials and methods) at 37° for 60 min and subjected to immunoblotting with anti-HA antibodies. Combinations of the following proteins and cofactors were used: E1, 4.5 μg Uba2p, 5.2 μg Aos1p; E2, 0.75 μg Ubc9p; E3, 4.5 μg Siz1pΔ440; ATP (10 mM) and 2.9 μg of 6xHis–Smt3p. The unmodified Top2p band is indicated by “u.” The Top2p–Smt3p conjugates are indicated with an asterisk. Multiple modified forms of Top2p were formed only in the presence of Smt3p.
Figure 3.
Figure 3.
Top2p tail is modified in vivo in a SUMO E3-dependent manner. (A) The COOH-terminal region of Top2p and the consensus sumoylation sites have limited conservation among yeast species. The S. cerevisiae Top2p tail region (residues 1219–1428) was aligned with the Candida albicans (C.g.) and Ashbya gossypii (A.g.) topoisomerases II and the secondary structure was predicted using the JPred package (Cuff and Barton 2000). h, alpha helix; e, beta-sheet. The SUMO consensus sites are marked with open boxes and numbered. The acceptor lysine residues are outlined. (B) Cell growth is not perturbed by the tagged top2 alleles ΔC and 3KR. Ten-fold serial dilutions from overnight cultures of wild-type (TOP2, TOP2:HA) and different top2 mutants were spotted onto YPD plates and incubated at the temperatures indicated. The tagged TOP2 allele replacement strains (W303-1A/pYT1033, W303-1A/pYT1034, and W303-1A/pYT1035) were generated after transformation with plasmids as described in materials and methods. The corresponding GFP-tagged strains (YPH499/pYT1026, YPH499/pYT1027, and YPH499/pYT1028) were also generated and showed no interference with cell proliferation or Top2p nuclear localization (data not shown). (C and D) Western blot analysis of Top2p sumoylation in vivo. Extracts from the strains 924-YPH499 (Siz+) or 924-4bAS399 (Siz−) expressing physiological levels of HF-Smt3p and carrying different top2 alleles were fractionated by IMAC. FT, flow through; EL, eluate. The wild type TOP2 gene and all top2 alleles were HA-tagged. HF–Smt3p conjugates eluted from the column were analyzed by Western blotting using anti-FLAG (C) and anti-HA (D) antibodies. The conjugated forms of Top2p (present only in Siz+ TOP2 cells) are indicated by an asterisk.
Figure 4.
Figure 4.
Epistatic interaction between the Smt3p-conjugation-deficient top2 mutations and SUMO E3 deficiency in the control of minichromosome stability. (A) Transmission efficiency of the pRS415 minichromosome. Minichromosome stability was measured (see materials and methods) at 30° in the wild-type (BY4733) and siz1/siz2 mutant (4bAS399) strains with different top2 variants (pYT1033, pYT1034, or pYT1035). (B) SUMO E3 mutants and top2 Smt3p-conjugation-deficient mutations stabilize dicentric minichromosomes. Transmission efficiency of pAS72, a conditional dicentric minichromosome, was measured at 30° in the wild-type (BY4733) and siz1/siz2 (4bAS399) strains with top2 variants (pYT1033, pYT1034, or pYT1035) as described in materials and methods.
Figure 5.
Figure 5.
Constitutive sumoylation results in pericentromeric targeting of Top2p. (A) Synthetic interaction between smt4-Δ and TOP3:SMT3 fusion. The same-concentration (106 cells/ml) cultures of three strains were plated on YPD plates in serial 10-fold dilutions and incubated at 30° (permissive for smt4-Δ) and 37° (nonpermissive for smt4-Δ) temperatures for 48 hr. Integrated TOP2 variants produce Top2p–HA fusions. SMT4 TOP2: W303 with the wild-type SMT4 gene transformed with pYT1033. smt4 TOP2 and smt4 TOP2:SMT3 are W303 with smt4 deletions, transformed with pYT1033 or pYT1051, respectively. As smt4-Δ results in massive lethality, even at permissive temperature, the starting dilutions have different number of growing colonies, as compared to Smt4+. (B) Layout of the PCR probes used for ChIP analysis of Top2p binding to the CEN4 region. (C) Smt3p fusion to Top2p tail results in Top2p enrichment at the CEN4 pericentromeric region. The W303 strains transformed with pYT1033 (TOP2:HA), pYT1051 (TOP2:SMT3:HA) or pYT1035 (TOP2-ΔC:HA), all replacing the wild-type TOP2 gene, were subjected to ChIP analysis using the PCR probes shown in B. ChIP analysis was as described (Strunnikov et al. 2001; Wang et al. 2004). (D–F) Fusion to Smt3p changes localization of Top2p–GFP in the nucleus. Spc42p-mRFP was used to mark SPB in diploid strains expressing both the wild-type Top2p and a corresponding Top2p–GFP fusion: TOP2:SMT3:GFP (D and E show maximal resolution) and TOP2-ΔC:GFP (E). Twenty optical Z-sections with 0.2-μm spacing were combined to compose the images (2 × 2 binning, 1-sec exposure per frame, except in E: no binning, 3-sec exposure per frame). Arrows point to clustered Top2p–Smt3p–GFP staining in mitotic cells.
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