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Is the yeast Anaphase Promoting Complex needed to prevent re-replication during G2 and M phases?

1997; Springer Nature; Volume: 16; Issue: 19 Linguagem: Inglês

10.1093/emboj/16.19.5988

1460-2075

Silke Pichler, Simonetta Piatti, Kim Nasmyth,

Genomics and Chromatin Dynamics

Article1 October 1997free access Is the yeast Anaphase Promoting Complex needed to prevent re-replication during G2 and M phases? Silke Pichler Silke Pichler Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Simonetta Piatti Simonetta Piatti Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria 26, I-20133 Milano, Italy Search for more papers by this author Kim Nasmyth Kim Nasmyth Research Institute of Molecular Pathology, Dr Bohr Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Silke Pichler Silke Pichler Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Simonetta Piatti Simonetta Piatti Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria 26, I-20133 Milano, Italy Search for more papers by this author Kim Nasmyth Kim Nasmyth Research Institute of Molecular Pathology, Dr Bohr Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Author Information Silke Pichler2, Simonetta Piatti3 and Kim Nasmyth1 1Research Institute of Molecular Pathology, Dr Bohr Gasse 7, A-1030 Vienna, Austria 2Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany 3Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria 26, I-20133 Milano, Italy The EMBO Journal (1997)16:5988-5997https://doi.org/10.1093/emboj/16.19.5988 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Anaphase Promoting Complex (APC) is required for anaphase progression and B-type cyclin proteolysis. The recent finding that inactivation of the APC allows 'over-replication' of DNA has led to the proposal that the APC might also be required for preventing reduplication of chromosomes during G2 and M phases. In this report we re-investigate the phenotype of apc mutant cells and find that they do not re-replicate their DNA during the period taken for wild-type cells to traverse G2 and M phases. apc mutants do, however, gradually increase their DNA content after long periods of cell cycle arrest. Such DNA synthesis occurs almost exclusively in the cytoplasm and neither occurs in cells lacking mitochondrial DNA nor depends on Cdc6, a protein which is essential for the initiation of chromosomal but not mitochondrial DNA replication. ARS1, a chromosomal replication origin, is not re-fired in cells deprived of APC function, confirming that the 'over-replicated' DNA in apc mutant cells is of mitochondrial origin. Furthermore, we find that APC function is required to promote but not to prevent re-replication in ndc10 mutant cells. We therefore propose that the APC is not involved in preventing re-duplication of chromosomes during G2 and M phases. Introduction Cells synthesize most of their constituents from instructions that reside within the DNA sequences of their chromosomes. They must therefore take inordinate care in duplicating this information and in segregating the two copies known as sister chromatids to opposite poles of the cell during mitosis. To maintain a stable karyotype, cells never—or only rarely—fire replication origins more than once between successive rounds of sister chromatid separation. It is not fully understood how they achieve this delicate feat. Recent work suggests that the initiation of DNA replication in eukaryotic cells is a two-step process, the first of which is the assembly of a large complex of 'initiator' proteins at future origins, known as pre-replication complexes (pre-RCs) (Diffley et al., 1994), and the second of which is the activation of S phase-promoting cyclin-dependent protein kinases, known as S-CDKs (Epstein and Cross, 1992; Schwob and Nasmyth, 1993; Jackson et al., 1995; Ohtsubo et al., 1995; Strausfeld et al., 1996; Krude et al., 1997). A related set of CDKs that are activated later during the cell cycle, known as M-CDKs, promote the formation of the mitotic spindle and the separation of sister chromatids (Booher et al., 1989; Moreno et al., 1989; Surana et al., 1991; Fitch et al., 1992; Basi and Draetta, 1995). In both budding and fission yeast, M-CDKs can also promote the initiation of DNA replication when S-CDKs are inactivated by mutation (Schwob and Nasmyth, 1993; Fisher and Nurse, 1996; Mondesert et al., 1996). S- and/or M-CDKs are active from the beginning of S phase through to the separation of sister chromatids at anaphase (for reviews see Nasmyth, 1993; Nurse, 1994). It has been proposed that S- and M-CDKs not only promote origin firing in cells that have previously formed pre-RCs but also prevent their de novo assembly (Dahmann et al., 1995). Thus, CDKs have opposing effects on the two steps of the initiation process, inhibiting the first step but activating the second. By this means, cells avoid entering a state in which they can both form pre-RCs and trigger origins that have formed them to initiate DNA replication. According to this hypothesis, each new round of origin firing depends on a cycle of CDK activity: a period of low activity that permits the assembly of pre-RCs followed by a period of high activity that permits origin firing. Re-firing of origins during S, G2 and M phases is therefore prevented by S- and M-CDK activity present during these phases of the cell cycle. A new round of replication depends on the inactivation of S- and MCDKs at anaphase. Budding yeast cells utilize two mechanisms to inactivate S- and M-CDKs during anaphase. First, they initiate ubiquitin-mediated proteolysis of B-type M-phase cyclins (Amon et al., 1994; Zachariae and Nasmyth, 1996) and second they turn off ubiquitin-mediated proteolysis of a potent CDK inhibitor called Sic1p (Schwob et al., 1994). Ubiquitination of B-type cyclins is mediated by an N-terminal destruction box (Glotzer et al., 1991) and depends on a large multisubunit complex called the Anaphase Promoting Complex (APC) or cyclosome (Irniger et al., 1995; King et al., 1995; Sudakin et al., 1995). The APC is involved in mediating the destruction, at anaphase, of many proteins besides cyclins, some of whose destruction may be necessary for the separation of sister chromatids (Cohen-Fix et al., 1996; Funabiki et al., 1996). Mutations in APC subunits prevent cyclin ubiquitination and degradation (Zachariae and Nasmyth, 1996). In several cases, such mutations also prevent the separation of sister chromatids at anaphase (Hirano et al., 1988; Irniger et al., 1995; Yamashita et al., 1996). The budding yeast APC has at least 10 subunits: Apc1p, Cdc16p, Cdc23p, Cdc26p, Cdc27p in addition to unidentified 150, 100, 80, 79 and 78 kDa proteins (Irniger et al., 1995; Zachariae et al., 1996; W.Zachariae, personal communication), homologues of which have been found in fission yeast (Hirano et al., 1990; Samejima and Yanagida, 1994; Yamashita et al., 1996), Aspergillus nidulans (Engle et al., 1990; O'Donnell et al., 1991), Xenopus laevis (King et al., 1995; Peters et al., 1996) and humans (Starborg et al., 1994; Tugendreich et al., 1995). It has recently been reported that mutations which inactivate the yeast APC and prevent both cyclin degradation and anaphase surprisingly do not prevent re-replication (Heichman and Roberts, 1996) as had previously been thought. This suggests that the APC, in addition to promoting anaphase, may be required to prevent re-initiation of DNA replication during G2 and M phases (Juang et al., 1997; for reviews see Heichman, 1996; Wuarin and Nurse, 1996). This hypothesis is interesting for at least two reasons. First, it implies that cells can after all assemble pre-RCs in the presence of high M-CDKs. Second, it implies that the APC has an as yet undefined role during G2 and M phases in preventing the accumulation of factors that promote origin firing. The degradation of such factors by the APC might normally depend on S- and M-CDKs, which would explain how these kinases also prevent re-replication during G2 and M phases. Alternatively, the hypothesis that CDKs inhibit assembly of pre-RCs might simply be wrong. Because this new finding about the APC promises to shed important new insight into the mechanism of re-replication control, we have re-investigated the phenotype of yeast apc mutants. We find that such mutants, upon being shifted to the restrictive temperature, do indeed eventually accumulate DNA. However, this DNA synthesis occurs largely if not exclusively in the cytoplasm, it fails to occur in rho0 strains lacking mitochondrial DNA, and it does not depend on the Cdc6 replication initiation protein. Furthermore, using a 5-bromodeoxyuridine (BrdU) incorporation assay capable of detecting replication of specific DNA sequences, we failed to detect DNA replication of even a small region surrounding the ARS1 origin of DNA replication. Our data suggest that the APC is not required to prevent re-replication of the nuclear genome during the G2 and M phases of wild-type cells. Results Cells deprived of APC function do not re-replicate during the period taken by wild-type cells to traverse G2 and M phases In previously published work characterizing DNA replication in a cdc23-1 apc mutant (Irniger et al., 1995), we isolated, by centrifugal elutriation, small unbudded daughter cells from a culture growing at 23°C and incubated them at 37°C. Using FACs analysis to measure cellular DNA content, we found that the mutant cells underwent a complete round of DNA replication at 37°C with kinetics that were similar if not identical to that of wild-type cells. They subsequently formed mitotic spindles but failed to separate sister chromatids. Under similar conditions, wild-type cells separated sister chromatids 30–40 min after the completion of DNA replication. The DNA content of cdc23-1 mutant cells remained constant (at 2C) for 120 min after completion of S phase but thereafter gradually increased. We obtained very similar data with cdc16-123 mutant cells (Figure 1A; for wild-type data see Zachariae et al., 1996, Figure 5) and in mutants lacking CDC26 (Figure 1B), which encodes a component of the APC that is essential only at 37°C and is required to prevent the dissociation of Cdc16p and Cdc27p from the rest of the complex (Zachariae et al., 1996; W.Zachariae, personal communication). Furthermore, all cdc26 deletion mutant cells (cdc26Δ) remained viable (that is, capable of giving rise to colonies of haploid cells) for 4 h after completion of DNA replication at 37°C (Figure 1C). In the case of cdc23-1 and cdc26Δ mutants, which clearly arrest at metaphase of the first cell cycle, few if any cells managed to separate sister chromatids during this period (Irniger et al., 1995; also data not shown). The key point is that we detected little if any DNA accumulation in cdc16-123, cdc23-1 or cdc26Δ mutants during the first 40 min after their completion of S phase; that is, during the interval taken by wild-type cells to traverse G2 and M phases. The gradual increase in the apparent DNA content of apc mutant cells after prolonged incubation could be due to mitochondrial DNA replication or indeed to an increase in non-DNA background fluorescence (both of which only become appreciable when cells become very large) or to re-initiation of rare origins. Heichman and Roberts (1996) observed more extensive DNA accumulation in cdc16 and cdc27 mutants released from a pheromone-induced G1 arrest at the restrictive temperature and proposed that it corresponds to re-replication of the nuclear genome. It is possible that the lesser DNA accumulation in our experiments (Figure 1A and B) is due to our having started with small cells that had not previously been cell cycle arrested. Figure 1.cdc16-123 and cdc26Δ mutant cells do not re-replicate DNA during the interval taken by wild-type cells to traverse G2 and M phases. Small daughter cells of cdc16-123 (K4103) (A) and of cdc26Δ (K7013) (B and C) were isolated by centrifugal elutriation (Time = 0) and incubated at 37°C in YEPD medium. (A and B) Budding index and DNA content were determined at the indicated time points. (C) Viability of cdc26Δ cells after incubation at the restrictive temperature for the indicated time periods. Download figure Download PowerPoint apc mutant cells gradually increase their DNA content after long periods of incubation at the restrictive temperature To investigate further whether apc mutant cells re-replicate their nuclear genome while arrested in metaphase at 37°C, we analysed the DNA content of cells after shifting asynchronous cultures from 23°C to 37°C. We found that cdc16-1 and cdc16-123 mutant cells accumulated with a 2C DNA content within 60 to 120 min but started to accumulate greater than 2C DNA contents after 180 min (Figure 2). This phenomenon occurred in both the strain background used by Heichman and Roberts (A364a) and our strain background (W303). We obtained similar results with cdc16-264 and cdc27-22 mutants (data not shown). Figure 2.Cells lacking APC function accumulate excess DNA after long periods of incubation at the restrictive temperature but fail to do so when mitochondria deficient. Rho+ and rho0 versions of cdc16-1 (KHY201, K6114) and cdc16-123 (K5600, K6823) strains were grown to logarithmic phase in YEPD medium at 23°C (cyc) and subsequently shifted to 37°C. After 3 and 6 h incubation at 37°C, the cultures were diluted 1:2 with prewarmed YEPD medium (37°C). At the indicated time points, samples were withdrawn for FACs analysis. Bg, background. Download figure Download PowerPoint The accumulation of excess DNA in cdc16 mutant cells occurs mainly in the cytoplasm To address whether the increasing DNA content of cdc16 mutants was due to re-replication of the nuclear genome, we monitored DNA synthesis within individual cells by immunodetection of BrdU incorporation (Neff and Burke, 1991). Thymidine kinase activity, which is required for BrdU labelling, was provided to the cells by introducing five copies of the thymidine kinase gene from herpes simplex virus under the control of the yeast GPD promoter [GPD-Tk construct; as described by Dahmann (1995)]. Asynchronous cultures of diploid wild-type and three different mutant strains were shifted from 23°C to 37°C for 2.5 h, after which they were incubated for a further 2.5 h at 37°C in the presence of BrdU. Most of the BrdU incorporation of wild-type cells was confined to regions of the cell that resemble the size and position of nuclei (it is not possible to localize BrdU incorporation and the position of nuclei in the same cells because the acid treatment needed to detect BrdU incorporation depurinates the DNA such that it can no longer be stained by DAPI). All wild-type cells contained staining consistent with replication of their nuclear DNA (Figure 3A–C). No staining was detected in control strains lacking GPD-Tk or when antibody against BrdU was omitted (data not shown). In contrast, the BrdU incorporation of cdc13-1 mutant cells, which are thought to arrest in M phase with a 2C DNA content, was distributed in small patches throughout the cell's cytoplasm and at the cell's periphery (Figure 3A). These patches presumably belong to mitochondria (see below). The pattern of BrdU incorporation of cdc16-123 and cdc16-264 mutants resembled that of cdc13-1 mutants (Figure 3A). Very few, if any, cdc16 mutant cells had nuclear staining similar to that of wild-type cells. The lack of nuclear staining was not due to dissolution of nuclei in the mutants because their nuclei were readily detected by DAPI staining of cells not treated with acid (Figure 3B). Figure 3.cdc16-123 and cdc16-264 mutant cells accumulate DNA mainly in the cytoplasm. Diploid wild-type cells carrying five copies of GPD-Tk (K6369) and congenic cdc16-123 (K6370), cdc16-264 (K6660), cdc13-1 (K6368) and esp1-1 (K6385) cells were grown to logarithmic phase in YEPD medium at 23°C (cyc) and subsequently incubated for 2.5 h at 37°C. The cultures were diluted 1:2 with prewarmed YEPD (37°C) and BrdU was added to a final concentration of 200 μg/ml. Cells were incubated in the dark for another 2.5 h at 37°C. (A) Cells were fixed after 2.5 h of BrdU labelling. Incorporated BrdU was visualized by indirect immunofluorescence using a monoclonal antibody specific for BrdU. (B) Cells were fixed and spheroplasted as for BrdU staining. DNA was visualized by indirect immunofluorescence using DAPI. (C) Samples for FACs analysis were taken at the indicated time points. DIC, differential interference contrast. Download figure Download PowerPoint To confirm that we were in fact capable of detecting re-replication using our BrdU incorporation assay, we performed the same experiment with an esp1-1 mutant, which is known to re-replicate the entire nuclear genome despite failing to undergo nuclear division (Baum et al., 1988). One-quarter or less of the esp1-1 nuclei accumulated with a 4C DNA content during the 5 h incubation at the restrictive temperature (Figure 3C) and a similar number of nuclei had nuclear BrdU staining (Figure 3A and B). Our assay is therefore clearly capable of detecting genuine re-replication. We conclude that very little nuclear DNA is replicated between 2.5 and 5 h after shifting cdc16 mutants to 37°C. The bulk of DNA synthesized during this period has a cellular distribution characteristic of mitochondrial DNA. apc mutant cells do not 'over-replicate' their DNA when mitochondria-deficient To investigate whether the DNA synthesized by cells lacking Cdc16 function belongs to mitochondria, we compared by FACs analysis the DNA accumulation of apc mutants containing mitochondrial DNA with congenic rho0 strains lacking it. We found that rho0 strains arrested permanently with a 2C DNA content. This result was obtained with cdc16-1, cdc16-123, cdc23-1, cdc27-22, cdc27-1 and apc1-1 mutants and, in the case of cdc16-1, in both the W303 and the A364a backgrounds (Figure 2 and data not shown). cdc27-22 was found to be a leaky mutation capable of going through multiple cell cycles before finally arresting in mitosis. Similar results were obtained when we repeated this experiment using medium containing 4% glucose (instead of the customary 2%); this was to test whether the failure of rho0 strains to synthesize excess DNA might be due to their less efficient utilization of glucose (data not shown). These data, like those obtained using BrdU staining, suggest that the bulk of the DNA synthesized by apc mutants shifted to the restrictive temperature is due to mitochondrial DNA. The chromosomal replication origin ARS1 does not re-fire in cdc16 mutant cells Our failure to detect appreciable BrdU incorporation within the nuclei of rho+ cells does not exclude the possibility that small but appreciable regions of the genome, for example those surrounding replication origins, might in fact be re-replicated in apc mutants. Our technique would probably not be sensitive enough to detect such small amounts of re-replicated DNA (10% of the genome or less). To investigate the possible re-replication of specific origin sequences, we used antibodies directed against BrdU to immunoprecipitate DNA containing BrdU and measured the quantity of specific DNA sequences in such immunoprecipitates using PCR (see Materials and methods). We used four sets of primers that amplified four different intervals in the region surrounding ARS1 (one of which contained ARS1 itself). Diploid cdc13-1, cdc16-123, cdc16-264, esp1-1 and wild-type (wild-type +Tk) cells containing GPD-Tk and wild-type cells lacking GPD-Tk (wild-type −Tk) were shifted to 37°C for 3 h and then incubated in the presence of BrdU for a further 2.5 h. All four fragments including ARS1 and its surroundings were amplified from the immunoprecipitates derived from wild-type and esp1-1 mutant cells expressing thymidine kinase from the GPD promoter but not from immunoprecipitates derived from wild-type cells lacking GPD-Tk (Figure 4). Using this technique, we failed to detect any incorporation of BrdU into ARS1 region sequences of cdc13-1, cdc16-123 and cdc16-264 mutants cells. These data suggest that ARS1, at least, does not re-fire in cdc16 mutant cells. Figure 4.ARS1 is not re-fired in cdc16 mutant cells. Congenic diploid cdc16-123 (K6370), cdc16-264 (K6660), cdc13-1 (K6368), esp1-1 (K6385) and wild-type (wild-type +Tk) (K6369) cells containing GPD-Tk and wild-type cells lacking GPD-Tk (wild-type −Tk) (K6380) were grown to logarithmic phase in YEPD medium at 23°C (cyc) followed by 3 h of incubation at 37°C. The cultures were diluted 1:2 with prewarmed YEPD medium (37°C), 200 μg/ml BrdU were added and cultures were incubated at 37°C for another 2.5 h (in the dark). (A) DNA was isolated and immunoprecipitated with antibody specific for BrdU. DNAs of immunoprecipitates (IP) and of whole cell extracts prior to immunoprecipitation (WCE) were subjected to PCR using four primer pairs to amplify ARS1 and three different surrounding sequences. The PCR products were separated by agarose gel electrophoresis. (B) At the indicated time points the cellular DNA contents were determined by FACs analysis. Download figure Download PowerPoint Figure 5.The continuous DNA synthesis in cdc16-123 mutant cells is not dependent on Cdc6p. Wild-type cdc6::hisG GAL-CDC6 (K6428), cdc16-123 CDC6 (K5600) and cdc16-123 cdc6::hisG GAL-CDC6 (K6433) strains were grown to logarithmic phase in YEPRafGal medium at 25°C (cyc) and subsequently shifted to 37°C for 2.5 h. Cells were harvested by filtration (Time = 0), resuspended in either YEPRafGal (Gal) or YEPD (Glc) medium (37°C) and incubated at 37°C for another 4 h. At the indicated time points, samples were collected for (A) Western blot and (B) FACs analysis. In (A) Swi6 was used as an internal loading control. Cdc6 was detected using the 12CA5 antibody. K699 (WT) was used as negative control for the 12CA5 antibody. Download figure Download PowerPoint Cdc6 is not required for the continuous DNA synthesis in cdc16 mutant cells Yet another way of addressing whether the increased DNA content of cells lacking APC function is due to nuclear DNA replication is to determine whether it depends on proteins, like Cdc6p, which are known to be required for chromosome duplication (Bueno and Russel, 1992; Kelly et al., 1993; Piatti et al., 1995). We therefore constructed wild-type and cdc16-123 strains, which were deleted for CDC6 and kept alive by an HA3-tagged version of the CDC6 gene driven by the GAL1,10 promoter (cdc6::hisG GAL-CDC6 strains). These strains proliferate normally in medium containing galactose, because the HA3-tagged Cdc6 protein is fully functional, but they fail to replicate their genomes and undergo a 'reductional' anaphase when shifted to medium containing glucose and lacking galactose, which represses transcription from the GAL1,10 promoter (Figure 5B; as described in Piatti et al., 1995). Wild-type and cdc16-123 mutant cells containing either a wild-type CDC6 gene or an HA-tagged version expressed from the GAL1,10 promoter (growing in the presence of galactose) were shifted to 37°C for 2.5 h and then transferred (still at 37°C) to a glucose-containing medium lacking galactose. Western blotting showed that the HA3-tagged Cdc6 protein disappeared within 30 min in both wild-type and cdc16-123 mutant cells (Figure 5A). FACs analysis, on the other hand, showed that the DNA content of cdc16-123 mutant cells gradually increased from 90 to 240 min after transfer to glucose medium, whether or not the cells contained Cdc6 protein (Figure 5B); that is, the gradual increase in the DNA content was similar if not identical in the CDC6 and cdc6::hisG GAL-CDC6 strains. Furthermore, the accumulation of DNA in cdc16-123 cdc6::hisG GAL-CDC6 cells transferred to glucose was similar to that of the same cells kept in galactose medium (Figure 5B). These data suggest that the accumulation of DNA in cdc16-123 cells incubated at the restrictive temperature is not dependent on the presence of Cdc6 protein. The Western data also suggest that proteolysis of Cdc6p is not mediated by the APC. This is an equally important point, because it has been suggested that the 'over-replication' of cdc16 mutants might arise due to their failure to degrade Cdc6p (Heichman and Roberts, 1996). Other data (Piatti et al., 1996) in fact suggest that Cdc6p proteolysis is mediated instead by the Cdc4/Cdc34/Cdc53/Skp1 ubiquitination system (known as the S phase Promoting Complex; for review see King et al., 1996). The re-replication of ndc10 mutant cells depends on APC function Ndc10p is a component of a kinetochore binding complex needed for the attachment of microtubules to chromosomes (Doheny et al., 1993; Goh et al., 1993; Jiang et al., 1993). Like esp1-1 mutants, ndc10-1 mutants fail to move sister chromatids to opposite poles of the cell during mitosis but nevertheless proceed to re-replicate their nuclear genome. If the APC had an important role in preventing re-replication during G2 and M phases, then one might expect that it would not be required for the re-replication of mutants that fail to undergo mitosis, such as ndc10-1 and esp1-1. If on the other hand, the APC were important for inactivating M-CDKs and thereby for generating a state of low CDK activity necessary for forming pre-replication complexes, then one might expect that the re-replication of ndc10-1 mutants would be abolished by mutating CDC16. According to the first hypothesis, cdc16-1 ndc10-1 double mutants should accumulate DNA in a pattern resembling ndc10-1 single mutants, whereas according to the second hypothesis, the double mutant should resemble cdc16-1 mutants in its pattern of DNA accumulation. We therefore compared, by FACs analysis, the pattern of DNA accumulation of cdc16-1, ndc10-1 and cdc16-1 ndc10-1 double mutants following their release (at 37°C) from a G1 arrest induced by mating pheromone (Figure 6). All mutants underwent a complete round of DNA replication within 80 min. About 50% of ndc10-1 cells underwent a second complete round between 120 and 200 min. Most if not all cdc16-1 mutant cells arrested initially with a 2C DNA content but thereafter gradually accumulated DNA, presumably of mitochondrial origin. The key point is that the pattern of DNA accumulation in the cdc16-1 ndc10-1 double mutant resembled that of the cdc16-1 single mutant. DNA accumulation occurred only gradually as in the cdc16-1 single mutant and not as a discrete doubling from 2C to 4C as in the ndc10-1 mutant. The gradual accumulation of DNA in cdc16-1 ndc10-1 double mutants was in fact somewhat more rapid than that seen in cdc16-1 single mutants, which could be due to the greater cell size of the double mutant cells and their greater number of mitochondrial DNAs. Very similar results were obtained when we compared accumulation of DNA in esp1-1 and cdc16-1 esp1-1 mutants (data not shown). These data demonstrate that the re-replication of ndc10-1 and esp1-1 mutants depends on APC function. Figure 6.Cdc16 function is needed for re-replication in ndc10-1 mutant cells. Wild-type (K699), ndc10-1 (K4610), cdc16-1 (K2529) and ndc10-1 cdc16-1 (SP365) cells were arrested by α-factor for 2.5 h at 25°C (Time = 0), collected by centrifugation, washed with YEPD medium (37°C) and then released into YEPD at 37°C. Samples for FACs analysis were taken every 30 min. Download figure Download PowerPoint Discussion APC function is needed for anaphase progression and cyclin destruction The Anaphase Promoting Complex (APC) is required for ubiquitin-mediated destruction of a number of proteins during mitosis. Its substrates in yeast include: B-type cyclins (Irniger et al., 1995); Pds1p and Cut2, that are inhibitors of sister chromatid separation (Cohen-Fix et al., 1996; Funabiki et al., 1996); Ase1p, a protein that is important for and associates with mitotic spindles (Juang et al., 1997); and Cdc5p, a protein kinase of the Polo family (M.Shirayama and K.Nasmyth, in preparation). The APC is composed of at least 10 different subunits (Engle et al., 1990; Hirano et al., 1990; O'Donnell et al., 1991; Samejima and Yanagida, 1994; King et al., 1995; Tugendreich et al., 1995; Peters et al., 1996; Yamashita et al., 1996; Zachariae et al., 1996; W.Zachariae, personal communication). Cells carrying temperature-sensitive mutations in APC subunits fail to destroy B-type cyclins, Pds1p, Ase1p and Cdc5p when shifted to the restrictive temperature. They also fail to separate sister chromatids and arrest in a metaphase-like state with intact mitotic spindles and high cyclin B/Cdk1 kinase activity (Irniger et al., 1995; Yamashita et al., 1996). Early work suggested that apc mutants also failed to enter a new cell cycle and to re-duplicate their chromosomes (Irniger et al., 1995), which fits with the current notion that M phase CDKs must be inactivated for chromosomes to be prepared for the next round of DNA replication (Broek et al., 1991; Hayles et al., 1994; Dahmann et al., 1995). A role for the APC in re-replication control? It has been recently proposed, however, that certain apc mutants accumulate large amounts of nuclear DNA during their cell cycle arrest with high M-CDK, which suggests that the APC might be required to prevent re-firing of origins during G2 and M phases (Heichman and Roberts, 1996). This proposal is of considerable interest, first, because it suggests that cells might after all be able to re-initiate DNA replication in the presence of high M-CDK activity and, second, because it suggests that the APC has an impo