Processing, localization, and requirement of human separase for normal progression

Anton Chestukhin*, Christian Pfeffer†, Scott Milligan†, James A. DeCaprio*, and David Pellman†‡

Departments of *Medical Oncology and †Pediatric Oncology, Dana–Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115

Edited by Joan V. Ruderman, Harvard Medical School, Boston, MA, and approved February 10, 2003 (received for review December 5, 2002) In all eukaryotes, anaphase is triggered by the activation of a separase forms a complex with , a inhibitor of the called separase. Once activated, separase cleaves a sub- separase activity (22–25). Release of the securin-mediated in- unit of , a complex that links replicated chromatids before hibition is achieved by anaphase-promoting complex-dependent anaphase. Separase and cohesin are conserved from yeasts to ubiquitination and degradation of securin (26, 27). In its turn, humans. Although the machinery for dissolving sister cohesion is activity of the anaphase-promoting complex is controlled by conserved, the regulation of this process appears to be more Mad2, a component of the mitotic checkpoint, which ensures complex in higher eukaryotes than in yeast. Here we report the that all kinetochores become attached to microtubules (reviewed cloning of full-length human separase cDNA and the characteriza- in ref. 28). The of securin is necessary for activation tion of the encoded protein. Human separase was observed at the of separase, but securin also appears to have a positive role in poles of the mitotic spindle until anaphase, at which time its facilitating separase activation (6, 29). Separase activity has association with the mitotic spindle was abruptly lost. The dynamic recently been shown to be inhibited by the cyclin-dependent pattern of localization of human separase during cell cycle pro- kinase Cdc2 (21). A third level of regulation occurs by the gression differs from that of fungal separases. Human separase also appears to undergo an autocatalytic processing on anaphase phosphorylation of the separase substrates. In yeast, phosphor- entry. The processed forms of human separase were isolated and ylation of Scc1 near the cleavage site by the Polo-like kinase the identity of the cleavage sites was determined by N-terminal Cdc5 facilitates separase cleavage (30). sequencing and site-directed mutagenesis. The processed catalytic The requirement for separase in dissolving sister cohesion has domain was found to be stably associated with the processed been directly demonstrated in yeasts by genetic analysis (8, 9, 11, N-terminal fragment. Finally, by depletion of endogenous separase 13, 31). An analogous role for separase in animal cells has been with antisense oligonucleotides, we report direct evidence that inferred from two lines of evidence. First, vertebrate Scc1 is a separase is required for high-fidelity separation in substrate of activated vertebrate separase. Second, tissue culture human cells. cells overexpressing a noncleavable form of human Scc1p fail to undergo sister chromatid separation (32). The hallmarks of this eparation of sister chromatids at anaphase requires precise defect in sister separation are the presence during anaphase Scoordination between cell cycle signals and the that of chromosomal bridges, multinucleated cells, and multipolar physically link replicated sisters. Chromatid cohesion is estab- spindles. lished during S phase and is maintained until onset of anaphase In this study, we directly characterized the role of human (1). The multisubunit protein complex that holds sisters together separase in . We find that human separase is associated is called cohesin (2–7). Scc1 subunit of the cohesin complex with centrosomes until anaphase, at which time spindle associ- undergoes a proteolytic processing at the -to- ation is abruptly lost. This event is correlated with the known anaphase transition, resulting in dissolution of the association timing of separase activation and the apparent processing of between sister chromatids (8). The cleavage of Scc1 appears to separase. By protein purification, microsequencing, and site- be both necessary and sufficient for initiation of anaphase and directed mutagenesis, we identify the sites of human separase pole-ward movement of (8–10). The specific and cleavage at anaphase. The sequence of the sites coupled with the highly regulated cleavage of Scc1 subunit is carried out by a analysis of a catalytically inactive human separase mutant sug- protease termed separase (9, 11, 12). In yeast, the entire gests that separase processing is autocatalytic. Finally, the role of pool of cohesin that is bound to sister chromatids (including the human separase in sister separation was directly tested by chromosome arms and centromeric regions) must be cleaved by depletion of separase with antisense oligonucleotides (ASO). separase to initiate anaphase (13). In vertebrates, most of the Together, these findings define novel and conserved features of cohesin dissociates from chromatids in prophase, before chro- the human that dissolves sister chromatid cohesion. matid separation in anaphase. A small fraction of cohesin remains in centromeric regions (10, 14). Dissociation of cohesin Materials and Methods from prometaphase chromosomes appears to be mediated by a Ј Polo kinase-dependent mechanism (15–17). However, both in Separase cDNA. The 5 -RACE PCR was performed by using yeast and vertebrates, complete cleavage of the chromosome- Marathon RACE kit, HeLa marathon-ready double-stranded Ј associated cohesin appears to be an essential prerequisite for (ds)cDNA (CLONTECH), and separase specific primers 5 - initiating anaphase. CCTCCAGCATCCAGACAACGGTAGATT-3Ј and 5Ј-GC- Separase belongs to the CD clan of cysteine (8). All CAAATCAACTATCTGACAG-3Ј. Major PCR products rang- members of this class share considerable homology within the ing from 850 bp to 1.4 kb were cloned into pCR-BluntII-Topo domain that contains the (18, 19). Another subclass of vector (Invitrogen) and sequenced (see also Fig. 5, which is CD clan endopeptidases are the (20). activa- published as supporting information on the PNAS web site, tion involves proteolytic processing of the proenzyme form to an www.pnas.org). activate processed form. Interestingly, what appear to be pro- cessed forms of separase were observed after metaphase when human separase is activated (10, 21). This paper was submitted directly (Track II) to the PNAS office. The mechanisms leading to separase activation are a major Abbreviation: ASO, antisense oligonucleotide. topic of current research. During the most of the cell cycle ‡To whom correspondence should be addressed. E-mail: David࿝[email protected].

4574–4579 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730733100 Downloaded by guest on September 24, 2021 Antibodies. A fragment of human separase corresponding to amino acids 1866–1966 was expressed in Escherichia coli as a GST fusion protein and used for immunization of rabbits (Babco, Richmond, CA). The same separase fragment was also expressed as maltose-binding protein (MBP) fusion protein by using pMal-c2X (New England Biolabs), purified, and coupled to CNBr-activated Sepharose 4B (Apbiotech͞Pharmacia). Im- mune serum was affinity purified on the column with immobi- lized MBP-separase as described (33). Monoclonal antiseparase antibodies XJ-13 were raised against the same C-terminal sepa- rase fragment used to generate the polyclonal antibody, with the exception that an MBP-separase fusion protein was used for immunization of mice instead of the GST-separase fusion.

Cell Lines. The human cervical carcinoma HeLa cell line was obtained from American Type Culture Collection (ATCC) and was cultured in DMEM (Mediatech, Herndon, VA) supple- mented with 10% Fetal Clone-I serum (HyClone), 100 units͞ml penicillin, and 100 ␮g͞ml streptomycin. Cell lines expressing either wild type of mutant separase were prepared from 293EcR cell line (Invitrogen). V5-epitope-tagged separase was inserted in pIND-V5–6His-Topo vector (Invitrogen). Transfection of the 293EcR cell line and selection of the positive clones on G-418 (0.4 mg͞ml) were carried out as described in the manufacturer’s protocol (Invitrogen). The cell lines were induced by Ponaster- one (Invitrogen) at a final concentration of 5 ␮M.

ASO Transfection. ASO transfection was carried out as described (34) with some modifications. Subconfluent HeLa cells were subjected to mitotic shake-off procedure to enrich in population ͞ of the cells in G2 M stage. The cells were allowed to attach for 2 h on six-well plates (Falcon) or glass coverslips and then were transfected with 500 pmols per well of various oligonucleotides and 10 ␮l per well of Lipofectamine 2000 reagent (Invitrogen). The separase-specific oligonucleotides (synthesized as phospho- thiorates to increase stability) are in Fig. 6A, which is published as supporting information on the PNAS web site. FITC-labeled random oligonucleotide was used as a tracer (10 pmols͞well), and scrambled oligonucleotide 7261 was used as a control.

Western Blot and Immunostaining. Western blot was performed according to ref. 33. For immunostaining, cells were cultured and transfected on glass coverslips. Cell staining was carried out as described elsewhere (35). Fig. 1. Autocatalytic activity of separase at multiple sites is required for posttranslational processing of the enzyme. (A) Addition of ponasterone- Results induced (؉) expression of V5-epitope C-terminally tagged C2029A mutant Autocatalytic Processing of Separase. The ORF for KIAA0165 separase or wild-type (wt) separase in 293 cells. Uninduced cells (Ϫ) were used ORF predicts a 1,795-aa polypeptide that shares substantial as a control. The C2029A mutant appears as a single polypeptide band homology with yeast orthologue of separase, Esp1 protein (36). corresponding to the full-length molecule. Expression of the wild-type sepa- We have identified an additional 5Ј-exon human separase by rase results in appearance of a 65-kDa cleavage product. The membrane was Ј probed with V5 antibodies. (B) Identification of the major cleavage site in 5 -RACE PCR. The full-length human separase cDNA encodes human separase. Expression of V5-epitope tagged wild-type separase was a protein of 2,121 amino acids (see for details, see Supporting induced for 14 h and immunoprecipitated with V5-tag antibodies. Ninety-five Results, which are published as supporting information on the percent of the precipitated proteins were separated by SDS͞PAGE, visualized PNAS web site). This cDNA was used to prepare the V5-epitope by Ponceau S staining (Preparative), or used for Western blotting with V5 tagged wild-type separase and other separase mutants used in antibodies. N-terminal sequencing analysis was performed on the 220-, 150-, this study. and 65-kDa protein bands, indicated by the arrows. Sequencing results are (Using the full-length separase cDNA, we characterized the given in the text. ؉ and Ϫ, induced and noninduced cells, respectively. (C posttranslational processing of human separase. We generated C-terminally epitope-tagged separase and mutant derivatives were tran- siently expressed in HeLa cells, and the whole-cell extracts were analyzed by inducible cell lines expressing either V5-tagged wild-type sepa- CELL BIOLOGY Western blot with V5 antibodies (see text for the description of the mutants). rase or a separase mutant that is predicted to be catalytically Full-length separase (arrow) and the proteolytic fragments resulting from inactive, C2029A (8, 32). The cysteine at the position 2029 of cleavage at each of the three sites are indicated (left). separase is essential for proteolytic activity and is conserved among all cysteine proteases (8). The separase cDNA or the C2029A mutant was expressed in 293 EcR cells under the control and 65-kDa (p65) polypeptides were present in apparently equal of the ecdysone promoter that is induced by ponasterone. We amounts. Both polypeptides were detected by an anti-V5 mono- found that the wild-type separase expressed 12–48 h postinduc- clonal antibody, suggesting that the full-length and truncated tion appeared as two polypeptide bands: a full-length protein forms both contain an intact C terminus. In contrast, induction (220 kDa) and a 65-kDa fragment (Fig. 1A). The 220-kDa (p220) of the C2029A mutant of separase expression resulted in the

Chestukhin et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4575 Downloaded by guest on September 24, 2021 appearance of only the full-length molecule; no cleavage prod- ucts were detected even on prolonged induction up to 72 h (data not shown). This finding suggests that separase processing may be autocatalytic because prolonged incubation of the C2029A separase in the presence of functional endogenous separase did not result in cleavage of C2029A separase.

Identification of Separase Cleavage Sites. Next, we used the induc- ible cell lines to identify the proteolytic sites in human separase. Large-scale immunoprecipitation by anti-V5 antibodies was performed by using extracts prepared from ponasterone- induced cells. Extracts prepared from the uninduced cells were used as a negative control. Most of the immunoprecipitation reaction was used for sequencing analysis, and a small fraction (Ϸ5%) was used for Western blot analysis with anti-V5-tag antibodies. The precipitated material was separated in SDS͞ PAGE and transferred to a poly(vinylidene difluoride) mem- brane. Ponceau S staining of the immunoprecipitated proteins revealed several polypeptide bands: a 220-kDa band correspond- ing to the full-length separase, a 150-kDa band, and the 65-kDa Fig. 2. Cellular localization of human separase: polyclonal antibodies. Shown band (Fig. 1B) (designated p220, p150 and p65, respectively). is immunofluorescence of mitotic HeLa cells, where separase-specific fluores- The intensity of the Ponceau S staining of all three bands was cence is red, ␥-tubulin is green, and DNA staining is purple. (A) Prophase HeLa cells similar. Western blot analysis demonstrated that p220 and p65 displays colocalization of separase (red) with ␥-tubulin (green), as evident from retained the C-terminal V5-epitope tag, whereas p150 was not superimposition of the images (yellow). (B) Ratio of the number of the cells detected by anti-V5 antibodies (Fig. 1B). containing separase labeling of centrosome to the total number of cells at the N-terminal sequencing of both p220 and p150 revealed that indicated stages of mitosis (n Ͼ 100 cells at each cell cycle stage). Note that these polypeptides start at position 1 of the full-length separase anaphase cells were scored as positive for separase even if the staining was fainter (1MRSFKRVNFGTL12). Identification of the intact N terminus than that observed for metaphase cells. The numbers therefore likely underes- timate the degree to which separase is lost from centrosomes at anaphase. (C) suggests that maturation of separase does not involve removal of Immunofluorescence images of prophase, metaphase, and anaphase in HeLa any N-terminal amino acids. Because p150 was not detected by cells showing separase, ␥-tubulin, and DNA staining. Shown are representative anti-V5 antibodies, we concluded that this fragment represents images from the analysis in B. a C-terminally truncated separase. Interestingly, p150 was spe- cifically immunoprecipitated by anti-V5 antibodies, suggesting that p150 forms a complex with V5-epitope-tagged forms of of these sites can be cleaved in vivo. For example, the Site 2 ϩ separase that contain the catalytic domain. 3 mutant was cleaved at the Site 1, whereas Site 1 ϩ 2 was cleaved The N-terminal sequence of the 65-kDa band revealed a full at Site 3 (although at apparently lower efficiency than at the Sites match with the internal separase sequence starting from 1507G 1 and 3; Fig. 1C). Quantitative Western blotting of the three (1507GSDGEDSASGXK1518). Importantly, the separase se- cleavage products (data not shown) suggests that the major quence preceding the cleavage site matches the consensus cleavage site was Site 2 (1501SFEILR2); however, if this site is sequence that has been deduced from the analysis of other lost, then cleavage at the other two sites becomes apparent. separase substrates, such as human Scc1 subunit of cohesin and Taken together, these data suggest that human separase, unlike 2 yeast Slk19 kinetochore-associated protein (SXEXXR ) yeast separase (29) undergoes autocatalytic processing. Further, (31, 37). our analysis of this process suggests that the full-length ORF, encompassing the new 5Ј-end exon reported here, is functional. Separase Contains Multiple Autocleavage Sites. In cohesin, the arginine residue N-terminal to the separase cleavage site is Separase Is Localized in Centrosomes Before Anaphase. Separase essential for cleavage (8, 32). Substitution of glutamic acid with family members Esp1 and Schizosac- arginine in the separase consensus sequence in human cohesin charomyces pombe Cut1 display spindle-pole localization during (for example EXXR to RXXE) blocks separase-mediated pro- mitosis. To study the subcellular localization of human separase, teolysis at this site (32). A separase mutant encoding the amino polyclonal antibodies raised against the C-terminal 1866–1996 acid substitutions E1503R and R1506E was generated (hereafter amino acid residue fragment of human separase were used for referred to as Site 2 mutant for the reasons described below). Transient expression of the Site 2 mutant in HeLa cells revealed indirect immunofluorescence. By Western blotting, our affinity that, like wild-type separase, it underwent autoproteolytic cleav- purified antibodies detected two major bands corresponding to age, suggesting the presence of additional autocleavage sites. full-length separase and to the processed C-terminal domain The slower electrophoretic mobility of the fragment resulting (see Fig. 6C). ASO depletion of separase confirmed that these from cleavage of Site 2 mutant relative to the wild type suggested two major bands correspond to the full-length and processed an alternative site upstream of the 1506R2G1507 site (Fig. 1C). forms of separase (see below and Fig. 6C). Preliminary cell Analysis of the separase sequence in the vicinity of the staining experiments suggested that human separase might 1506R2G1507 revealed two additional potential cleavage sites: localize to the centrosome (data not shown). To test this ␥ 1481GPEIMR2, and 1530EWELLR2. To determine whether possibility, double labeling was performed by using anti- - these were genuine separase cleavage sites, these sites were tubulin antibodies to identify centrosomes. The double-labeling mutated by double-point substitutions E1483R and R1486E immunofluorescence experiments in HeLa (Fig. 2 A and C), (Site 1) and E1532R and R1535E (Site 3). In addition to the A549 and U-2 OS (data not shown) cells revealed colocalization mutations of the single sites, mutants altered at either two or all of separase and ␥-tubulin in centrosomes during the early stages potential cleavage sites were generated. Western blot analysis of of mitosis. In prometaphase and metaphase, the majority of cells HeLa cells expressing the Site 1 ϩ 2 ϩ 3 mutant failed to detect (65.2% and 73.7%, respectively, n Ͼ 100) displayed two separase any specific cleavage products (Fig. 1C). This suggests that any foci that were identified as the centrosomes based on colocal-

4576 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730733100 Chestukhin et al. Downloaded by guest on September 24, 2021 Fig. 3. Detection of endogenous human separase: monoclonal antibodies. (A) Whole-cell extracts were prepared from control 293 cells (Ϫ) or the cells induced to express recombinant V5-tagged full-length separase (ϩ). Identical panels were probed with either V5-tag-specific (V5) or separase-specific (XJ-13) mAbs. (B) Endogenous separase was detected in 293 cells by using XJ-13 mAb and a TRITC-labeled secondary Ab, ␥-tubulin was stained by polyclonal Ab, and Cy2-labeled secondary Ab and DNA was visualized by 4Ј,6-diamidino-2-phenylindole staining. pm, m, a, and t, prometaphase, metaphase, anaphase, and telophase cells, respectively.

ization with ␥-tubulin (Fig. 2B). Strikingly, as the cells pro- Depletion of Endogenous Separase Results in Increase of Aberrant gressed through anaphase, the separase-specific signal was lost Mitosis. A key role for separase as the trigger for anaphase entry (Fig. 2 B and C). Only 10% of the early and late anaphase cells in mammalian cells was suggested by the observation that displayed either one or two foci, and no foci were observed in overexpression of a noncleavable cohesin subunit Scc1 results in cells in telophase or during cytokinesis (Fig. 2B, n ϭ 100). The abnormal mitosis (32). To directly determine whether separase loss of separase signal from the centrosomes was correlated with was required for normal mitosis, we depleted endogenous hu- the proposed timing of separase function at anaphase onset (38). man separase by use of ASO. A series of various 18-mer We did not detect the redistribution of separase from the oligonucleotides were synthesized and tested for their ability to centrosomes onto mitotic spindles, as was observed for the yeast reduce steady-state level of endogenous separase. HeLa cells separase (8, 38, 39). were transfected with the indicated ASOs and relative amounts To verify the specificity of the centrosomal localization of of separase were compared by Western blot by using antisepa- human separase, we prepared a monoclonal antibody against the rase antibodies (Fig. 6B). Although several ASOs resulted in C-terminal fragment of human separase (residues 1866–1996). depletion of separase, the most consistent depletion of the endogenous enzyme was achieved with oligonucleotide 7106. To This mAb (designated XJ-13) recognized both the full-length optimize conditions further, the ASO 7106 was tested at various and autoprocessed forms of recombinant and endogenous sepa- concentrations from 30 to 500 nM. We found that depletion of rase (Fig. 3B). Of note, by Western blotting, the XJ-13 mAb was separase was dosage-dependent and that maximal depletion was A highly specific (Fig. 3 ). By indirect immunofluorescence, XJ-13 achieved at 500 nM. Further increase of the concentration of the gave the identical pattern of centrosomal labeling as we observed ASO 7106 did not result in decrease of the separase signal (data for the affinity-purified polyclonal antibodies. Colocalization of not shown). As a control, a scrambled oligonucleotide with the separase with ␥-tubulin was observed in 95% of prometaphase ϭ ϭ same nucleotide composition as 7106 was synthesized and des- cells (n 110) and in 98% of metaphase cells (n 120), whereas ignated 7621. CELL BIOLOGY only 2% of anaphase cells displayed chromosomal localization of To evaluate the effect of separase depletion on the segregation ϭ separase (n 105). The centrosome-specific labeling of cells by of sister chromatids, we performed immunostaining of the cells XJ-13 mAb was blocked by the preincubation of the mAb with transfected with either 7106 ASO or the control oligonucleotide the excess antigen (see Fig. 7A, which is published as supporting 7621. Transfection efficiency in the ASO experiments (estimated information on the PNAS web site). Furthermore, the centro- by cotransfection of an 18-mer fluorescent oligonucleotide) was some-specific labeling of cells by XJ-13 was dramatically reduced consistently in the 90–95% range (data not shown). To verify the (from 98% to 30%, n ϭ 200) in prometaphase and metaphase efficiency of ASO-specific separase knockdown, Western blot cells transfected with a separase-specific ASO (see below and analysis of the extracts prepared from transfected cells was Fig. 7B). performed (Fig. 4A). We found that transfection with 500 nM

Chestukhin et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4577 Downloaded by guest on September 24, 2021 rial was detectable between segregated DNA masses were scored as aberrant (Fig. 4C, Aberrant). The frequency of abnormal mitosis in cells transfected with oligonucleotides 7106 vs. 7621 is shown in Fig. 4B. Antisense oligonucleotide-mediated depletion of separase results in a Ͼ4-fold increase in the mitotic cells with lagging chromosomes. This finding directly demonstrates that separase is required for normal chromosome segregation. Discussion The separase system controls the timing of anaphase onset in all eukaryotes (3, 4, 6, 40, 41). Although the components for sister chromatid separation are conserved, there are important species differences that may be responsible for species-specific regula- tion. In this paper, we characterize human separase. Our findings emphasize the basic conservation of the system but highlight potentially important unique features of human separase. The localization of human separase has both similarities and differences from the previously reported localization of yeast separases. Both budding and fission yeast separases localize to the centrosome and to the mitotic spindle (8, 38, 39). In yeast, separase localizes to both the centrosome and spindle either before anaphase (fission yeast) or at anaphase onset (budding yeast) and then persists on the spindle until midanaphase. Using affinity purified polyclonal antiseparase Abs and an antiseparase mAb XJ-13, we have demonstrated that human separase local- izes to the centrosomes. We have not observed separase staining along spindle microtubules. Unlike yeast separase, human sepa- rase was observed on centrosomes͞spindle poles only before anaphase and then was abruptly lost on anaphase onset. Al- though we cannot exclude the possibility of a spindle-associated pool of separase that is below our detection levels, these findings suggest that human separase may not have the crucial role for maintaining anaphase spindle integrity that has been observed for yeast separase (8, 37–39). The localization of human separase to the centrosome is potentially important, because Caenorhab- ditis elegans separase was recently shown to be required for migration and cortical association of the paternal centrosome after fertilization. This process is crucial for the establishment of normal anterior–posterior polarity of the embryo (42). More- over, separase is also required in budding yeast for establishing normal spindle position (43). Our identification of three cleavage sites in human separase has potential implications for the mechanism of separase acti- vation and regulation. Unlike wild-type separase, a catalytically inactive human separase was not processed in cells containing active endogenous separase (Fig. 1A). The simplest interpreta- Fig. 4. Transfection with various antisense oligonucleotides results in de- tion of this result is that the processing of human separase is pletion of separase. (A) HeLa cells were transfected with 7106 and control 7621 autocatalytic. It is interesting to note that Drosophila separase oligonucleotides at a final concentration of 500 nM. Twenty-four hours after appears to be encoded by two separate , one encoding a transfection, the cells were harvested in sample buffer and separated in small protein containing the catalytic domain and another that SDS͞PAGE. The steady-state levels of separase were compared by Western blot may function analogously to the N terminus of human separase by using antiseparase polyclonal antibodies. V5-Sep., the lane where trans- (44). By contrast, budding yeast separase does not appear to be fected V5-tagged human separase was loaded as a control. After separase processed. However, the N terminus of budding yeast separase detection, the membrane was stripped and reprobed with anti-Vinculin an- was recently shown to interact with the C-terminal catalytic tibodies (Vin.), confirming the equal loading of the samples. (B) The relative numbers of cells undergoing anaphase after transfection with oligonucleo- domain, explaining why the extreme N terminus is absolutely tides 7106 or 7621. The ratio of abnormal bridging chromosomes to the total required separase activity (29). Here we demonstrate that the number of mitotic cells was calculated by counting Ͼ500 mitotic cells from N-terminal 150-kDa fragment of human separase is tightly three independent experiments. (C) Examples of normal and abnormal an- associated with the processed catalytic domain. Thus, despite aphase figures from the experiment described in B. MB indicates midbody, some species differences, an interaction between the N- and and CB indicates a chromosomal bridge. C-terminal separase domains may be a common theme for all separases. Although we think it is likely that separase processing follows the initial activation of separase, processing may be 7106 ASO resulted in a 10- to 50-fold reduction in separase important to stabilize the catalytically active conformation or for signal. Immunostaining with antitubulin antibodies was per- regulation. formed to distinguish mitotic and postmitotic cells from total cell While this manuscript was under review, two studies were population. Fig. 4C (Normal) shows mitotic cells in anaphase published that independently demonstrated the autocatalytic that were scored as normal as evident by the absence of lagging processing of human separase and the physical association of the chromosomes, whereas cells where lagging chromosomal mate- N- and C-terminal separase fragments (45, 46). Waizenegger et

4578 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730733100 Chestukhin et al. Downloaded by guest on September 24, 2021 al. (45) identified potential separase autocleavage sites based on (10, 14). Previous work has established that human separase can the known consensus sequence and by transient expression of cleave the Scc1 subunit of human cohesin (10). Furthermore, potential cleavage site mutants in HeLa cells. Zou et al. (46) noncleavable mammalian Scc1 interferes with sister separation identified the same sites by peptide sequencing of processed (32). Although these experiments supported the idea that mam- separase transiently expressed in 293T cells. Both groups found malian separase has essentially the same role in separating sisters that a separase mutant lacking all three cleavage sites was not as yeast separase, an alternate interpretation of these results was detectably compromised for proteolytic activity against the Scc1 that the defect in sister separation was due to an indirect subunit of cohesin (45, 46). Additionally, Zou et al. (46) found consequence of overexpressing the noncleavable variant of Scc1 that overexpressed functional separase can induce the processing rather than a direct effect of failing to cleave cohesin. Here we of catalytically inactive separase, suggesting that at least some observe a virtually identical effect of separase depletion on sister separase processing can occur by an intermolecular reaction. We separation as that reported for cells expressing noncleavable did not detect processing of catalytically inactive separase in the Scc1 (32), strongly supporting the model that separase activation presence of functional endogenous separase. The intermolecular at anaphase in mammalian cells is the primary trigger for sister reaction may therefore be inefficient, raising the possibility that chromatid separation. a significant amount of separase processing could occur by an intramolecular reaction. We thank Larisa Litovchick for fruitful discussions and help in prepa- Our depletion of human separase with antisense oligonucle- ration of the manuscript. This research was supported in part by National otides provides direct evidence for a crucial role for separase in Research Service Award 1F32 CA84752 and a research grant to A.C. separating sister chromatids in human cells. The possibility that from the Elsa U. Pardee Foundation (Midland, MI). J.A.D. is a Scholar the mechanism of sister chromatid separation might differ in of the Leukemia and Lymphoma Society, supported by National Insti- some respects between yeast and mammalian cells was raised by tutes of Health Grant RO1-CA63113. D.P. is a Scholar of the Leukemia the finding that the bulk of mammalian cohesin dissociates from and Lymphoma Society and is supported by the National Institutes of chromosomes well before sister separation at anaphase onset Health Grant RO1 CA55772.

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