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© The Rockefeller University Press,
0021-9525/1997//345 $5.00
The Journal of Cell Biology, Volume 136, Number 2,
, 1997 345-354
Article |
Identification of a Mid-anaphase Checkpoint in Budding Yeast
Activation of a facultative, dicentric chromosome provides a unique opportunity to introduce a double strand DNA break into a chromosome at mitosis. Time lapse video enhanced-differential interference contrast analysis of the cellular response upon dicentric activation reveals that the majority of cells initiates anaphase B, characterized by pole–pole separation, and pauses in mid-anaphase for 30–120 min with spindles spanning the neck of the bud before completing spindle elongation and cytokinesis. The length of the spindle at the delay point (3–4 µm) is not dependent on the physical distance between the two centromeres, indicating that the arrest represents surveillance of a dicentric induced aberration. No mid-anaphase delay is observed in the absence of the RAD9 checkpoint gene, which prevents cell cycle progression in the presence of damaged DNA. These observations reveal RAD9- dependent events well past the G2/M boundary and have considerable implications in understanding how chromosome integrity and the position and state of the mitotic spindle are monitored before cytokinesis.
Abbreviations used in this paper: DE and VE, digital and video enhanced; DIC, differential interference contrast; FISH, fluorescent in situ hybridization.
Budding yeast has been one of the key genetic systems in defining the progression of cell cycle events. One of the major paradigms is the accumulation and destruction of cyclin–cyclin-dependent kinase complexes (Nasmyth, 1993; Murray, 1995a). By delaying or advancing the timing of cyclin–cyclin-dependent kinase activity, the cell is able to coordinate steps in cell cycle progression, as well as respond to environmentally induced damage before downstream events. Cells can delay the onset of DNA synthesis (S phase) or mitosis (M phase) after exposure to x-rays, 
irradiation, or ultraviolet light (Hartwell and Weinert, 1989; Weinert and Lydall, 1993; Murray, 1995b). DNA lesions are recognized by proteins engaged either in DNA processing (Rad9, 24, 17, and Mec3) or signalling (Mec1 and Mec2/Rad53) (Siede et al., 1993; Allen et al., 1994; Weinert et al., 1994; Lydall and Weinert, 1995). Aberrant spindle assembly and alterations in kinetochore structure and function are also subject to surveillance mechanisms that act to delay anaphase onset (Hoyt et al., 1991; Li and Murray, 1991; Spencer and Hieter, 1992; Gorbsky and Ricketts, 1993; Li and Nicklas, 1995; Wang and Burke, 1995). These lesions are monitored, at least in budding yeast, by the MAD1, 2, and 3 and BUB1, 2, and 3 genes (Hoyt et al., 1991; Li and Murray, 1991; for review see Wells, 1996).
Relatively little information addresses cell cycle regulation and checkpoint pathways that may be critical for coordinating spindle position and disassembly with cytokinesis. Our recent results from high-resolution video enhanced- (VE)1 and digital enhanced- (DE) differential interference contrast (DIC) microscopy of living cells reveal discrete kinetic and morphological transitions during anaphase progression (Yeh et al., 1995). Most notable are the sequential fast and slow phases of spindle elongation and the morphological transition from a sausage shape to a bi-lobed nucleus that accompany these rate changes. At anaphase onset, the 1.5–2 µm spindle spanning the preanaphase nucleus elongates rapidly through the neck of the budded cell (1.0 µm/min) until the spindle and nucleus achieve a length of 
3–4 µm in a haploid cell. The rate of spindle elongation decreases (0.3 µm/min), and the nucleus converts from a sausage-shaped structure to a bi-lobed configuration and elongates until the maximal length of 10–12 µm is reached. Similar rates and evidence for biphasic spindle elongation have been obtained from observations of spindle pole body movement in living cells, as well (Kahana et al., 1995). Chromatin separation, as seen by staining with DAPI, appears to be completed in the bi-lobed nucleus. Differential regulation of microtubule dynamics and nuclear and cytoplasmic motor proteins are likely to be involved in the regulation and translocation of the spindle during anaphase. Indeed, spindle elongation is confined primarily to the mother in cells lacking the microtubule-based motor protein dynein (DHC1), and spindle disassembly and cytokinesis are delayed until the nucleus traverses the neck of the budded cell (Eshel et al., 1993; Li et al., 1993; Yeh et al., 1995). The spatial consequence of division by budding is that the plane of cleavage is established well before bi-polar spindle formation. This configuration requires spindle migration through the neck before successful cell division, and the dynein mutants reflect the cellular capacity to monitor spindle and/or nuclear position.
Studies in populations of budding yeast, after activation of a facultative, dicentric chromosome are also indicative of a mid-anaphase cell cycle checkpoint which can delay progression through mitosis (Neff and Burke, 1992; Brock and Bloom, 1994). These dicentric chromosomes contain two centromeres, one of which is conditionally regulated by the GAL1 transcriptional promoter (Brock and Bloom, 1994). Transcription is repressed on glucose, and the centromere is completely functional. Growth on galactose activates the promoter, which in turn, inactivates the centromere. The conditional dicentric chromosome is stably maintained in a monocentric state by growth on galactose and becomes dicentric on glucose. Cells harboring an active, dicentric chromosome are delayed in their cell cycle transit. A third to one half of the cells in a population are large budded, with nuclear DNA spanning the neck and a short spindle bisecting the nucleus, after the switch to glucose as the sole carbon source for growth (Neff and Burke, 1992; Brock and Bloom, 1994). p34cdc28 kinase activity is elevated in cells containing an active dicentric chromosome, compared to cells with monocentric chromosomes grown on glucose for comparable times (Neff and Burke, 1992; Brock and Bloom, 1994). The inference from these studies was that the delay preceded the decline of p34cdc28 kinase activity, and the cells were arrested prior to the exit from mitosis.
In the present study, we have used video enhanced and digital enhanced-DIC microscopy to determine the kinetics of spindle elongation in individual cells harboring an active dicentric chromosome. Having established a framework of morphological landmarks for anaphase spindle progression in wild-type cells (Yeh et al., 1995), we have quantitated the kinetics of anaphase, spindle morphology, and spindle length, in individual cells containing active dicentric chromosomes from anaphase to cytokinesis. These studies clearly demonstrate a mid-anaphase delay which is dependent upon the RAD9 checkpoint gene. RAD9 has been shown previously to prevent cell cycle progression in the presence of DNA damage prior to anaphase onset (Weinert and Hartwell, 1988). Our results are indicative of a regulated progression from early anaphase to late anaphase during the later stages of mitosis.
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Strains and Plasmids
All strains used in this study are isogenic derivatives of J178-1D (Mat a ade1 met14 ura3-52 leu2-3,112 his3). The 45-kb dicentric chromosome III strain J178#24 (his4
::URA3 GALCEN3) was constructed by fragment-mediated transformation of the conditional centromere at the HIS4 locus (Hill and Bloom, 1989). The rad9 dicentric strain was constructed by fragment-mediated transformation into J178#7 (his4::URA3 GALCEN3:: his4), as previously described by Brock and Bloom (1994). The 3-kb dicentric plasmid yC2 (Hill and Bloom, 1987) was introduced into J178-1D by standard transformation. All dicentric strains were grown in galactosecontaining medium to maintain chromosome III or the plasmid yC2 as monocentric.
Immunofluorescence
Immunofluorescent staining of yeast cells was performed essentially as described by Pringle et al. (1989). Cells were grown to early to mid-logarithmic phase growth in galactose-containing medium. Cells were pelleted, washed once with sterile water, resuspended in glucose-containing medium, and returned to 32°C for 1–2 h. Cells were fixed in 3.7% formaldehyde for 2 h at 25°C before processing for immunofluorescence. Microtubules were visualized with the rat anti–
-tubulin antibody YOL1/34 (Accurate Chemical and Scientific Corp., Westbury, NY). Rhodamine-conjugated goat anti– rat (Cappel Labs, Malvern, PA) was used as the secondary antibody. Cells were incubated with DAPI (1 µg/ml) for 5 min before the addition of mounting medium. Images were acquired and stored for further analysis, as described for FISH. Fluorescent digital images were analyzed with distance measuring tools within Metamorph software (Salmon et al., 1994).
To visualize the nucleus, cells were fixed in 3.7% formaldehyde for 1 h at 25°C. Fixed cells were washed once with water and resuspended in 1 µg/ ml DAPI for 5 min. The cells were washed once with water and mounted in mounting medium.
Fluorescent In Situ Hybridization
J178#24 was grown to an A660 = 0.2 density in galactose-containing medium before switching to YPD for 1 h. Cells were fixed and stained with DAPI (1 µg/ml for 5 min) or subjected to fluorescent in situ hybridization (FISH) as described by Guacci et al. (1994). Images were visualized with a microscope (Microphot FXA; Nikon) using a 60X/1.4NA PlanApo objective. Metamorph software (Universal Imaging Corp., West Chester, PA) was used to control a Metaltek filter wheel (Salmon et al., 1994). A shuttered mercury lamp was used for fluorescence excitation (490 nm). Images were collected with a cooled CCD camera (C4880; Hamamatsu) and stored on an optical drive (Pinnacle Micro, Irvine, CA). 100–200 nuclei were counted.
Real Time Analysis of Activated Dicentric Chromosomes
Cultures were grown to early logarithmic growth phase and spotted directly from galactose medium to YPD/25% gelatin slabs. Cells were filmed using high resolution VE–DIC and DE–DIC microscopy and analyzed as described in Yeh et al. (1995).
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5 ± 1 min (Fig. 4 A). As the spindle elongated to 4 µm in length, the nucleus remained rounded in shape as it traversed the neck of the dividing cell (Fig. 2 C). The time from nuclear protrusion to the formation of this rounded sausage nucleus took 10 ± 6 min (Fig. 4 A). Interestingly, the nucleus continued to maintain this round to sausageshaped morphology for 47 ± 18 min before further spindle elongation and the transition to bi-lobed structures. This is significantly longer than the 9 ± 2 min transition in wildtype cells, indicating a significant delay in both the kinetics and morphological transitions that accompany anaphase B. Once the bulk of the nucleus formed a bi-lobed morphology and began the slow phase of spindle elongation, the time to cytokinesis was 38 ± 10 min, similar to that observed in wild-type cells.
Coincidence of Nuclear and Spindle Position at the Mid-anaphase Delay
Bending of the mitotic spindle in and out of the plane of focus often precludes direct visualization of the spindle by VE-DIC microscopy. To confirm that position of the nucleus during mid-anaphase corresponds to actual spindle elongation through the neck, we compared the length of the nucleus in fixed cells, as determined by DAPI staining, to the length of the mitotic spindle, as visualized by indirect immunofluorescence. Cells containing the conditional dicentric chromosome were grown on galactose-containing medium and switched to glucose-containing medium for 2 h to activate the dicentric chromosome. Cells were fixed and stained for nuclear (DAPI) and spindle (anti–tubulin immunofluorescence) analysis. In all budded cells with DNA spanning the neck (36/36), the mitotic spindle had elongated through the neck (compare Fig. 5, C and D; E and F). Quantitation of the length of tubulin immunofluorescence and of DAPI staining along the mother bud axis in G2 and mid-anaphase arrested cells shows an excellent correlation between the two measurements. In G2 cells (medium size bud, nucleus near the neck), the mean spindle length was 1.25 ± 0.23 µm, and the mean nuclear length was 1.31 ± 0.19 µm (n = 13) (Fig. 5, A and B). Early stages of anaphase in cells containing an active dicentric chromosome were often characterized by finger-like protrusions of the nucleus into the bud (Fig. 2 B, VE-DIC; Fig. 5 D, DAPI staining). Correspondingly, the spindle spanned the distance from the end of the bulk of chromatin in the mother to the tip of the finger-like protrusion in the bud (Fig. 5 C, 2.54 µm in length). In cells exhibiting a mid-anaphase pause, the mean spindle length was 4.17 ± 0.92 µm and the mean nuclear length was 4.09 ± 0.91 µm (Fig. 5, E and F, n = 31). Thus the fixed images corroborate the progression, as well as the length measurements, in early to mid-anaphase, as determined in the real time analysis, and indicate the close correspondence between nuclear and spindle elongation throughout anaphase.
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22 min (Fig. 3 B). Cells delayed in anaphase B progression (Fig. 4 B) displayed the initial rapid elongation phase (2–4 µm), which was followed by a pause in spindle elongation in which the nucleus remained in the neck for a significant amount of time (up to 60 min in Fig. 4 B). Only after this pause, was the second phase of elongation able to proceed to the maximal spindle length of 10–12 µm.
Sister Chromatid Separation at the Mid-anaphase Transition
The partially elongated spindles indicate a delay in anaphase B spindle pole separation. However, the degree of chromosome separation was not apparent in the VE-DIC images. We therefore used FISH to visualize sister chromatids in cells arrested in mid-anaphase. Asynchronous populations of cells containing the conditional dicentric chromosome were grown on galactose and switched to glucose for 1–2 h, when 16% of the cells were arrested as large budded cells with DNA spanning the neck (Table I).
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9 min (Fig. 3 A). 9/90 min doubling time predicts that 10% of the population will be caught in the mid-anaphase stage. This value compares quite favorably to the 8% determined by FISH (Fig. 6 B; Guacci et al., 1994). By the same reasoning we can predict what fraction of cells harboring a dicentric chromosome will be in mid-anaphase in a logarithmic growing population. The dicentric delay persists for 57 min (Fig. 4). The doubling time of this population is 150 min, resulting in 38% of the cells at the midanaphase transition at any one time. However, only about a third of the cells in a population pause in response to activation of the dicentric chromosome (Neff and Burke, 1992; Brock and Bloom, 1994). We therefore estimate 2/3(8%) + 1/3(38%) = 17.8% of cells to be in mid-anaphase. This value compares quite favorably to the 16% of cells found to be in mid-anaphase, as determined by DAPI staining (Table I), but less than that determined by FISH (34%). While the high FISH value is certainly indicative of sister chromatid separation in the population of mid-anaphase cells, it may also reflect centromere separation in a fraction of large-budded cells with DNA at the neck (Table I) as well as cells with separated nuclei (Table I, last column), in which the spindles may have collapsed and given rise to apparently undivided nuclei with two spots. In any case, the centromere regions of sister chromatids have separated in nuclei of cells containing active dicentric chromosomes, indicating these cells traverse the metaphase/ anaphase boundary and become blocked following either anaphase A or anaphase A and B, with partially elongated spindles, i.e., mid-anaphase.
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15 µm in its linear form. Estimation of the packing ratio in chromatin (sevenfold around a nucleosome) or the degree of condensation from FISH (80 fold; Guacci et al., 1994) provide an approximate length of this region to be anywhere from 0.2–2 µm in vivo. Similarly, dicentric chromosomes with their centromeres 3 kb apart would be expected to span 0.02–0.2 µm in vivo. If the physical constraint model is correct, the length of the spindle at the arrest point would reflect centromere–centromere distances, and activation of a dicentric chromosome with the centromeres 3 kb apart might result in a 10-fold difference in spindle length at the delay point. As shown in Fig. 8, cells containing the 3-kb dicentric minichromosome exhibit similar kinetics and morphologies as those of a cell containing the 45-kb dicentric chromosome. Thus the midanaphase pause is not a function of the length between the two centromeres.
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There are several aspects of dicentric chromosome activation that may contribute to the delay. One is that tension generated by the chromosome bridge could restrain spindle elongation. The finger-like protrusions of the nucleus into the bud (Figs. 4 A and 5 D), appear to be futile attempts by the spindle/nucleus to initiate anaphase spindle pole separation. This period (5 min duration) has no correlate in the kinetic progression of a wild type cell. These protrusions, and the rounded morphology of the nucleus as it traversed the neck of the dividing cell (Fig. 2 C), probably reflect transient physical constraints on the spindle. The time lapse studies with centromeres at different distances (3 vs. 45 kb apart) indicate that the arrest in spindle elongation is not exclusively due to a DNA bridge constraining anaphase pole separation. Cells containing either dicentric chromosome display virtually identical spindle morphologies and lengths (4 µm) for similar durations (47–59 min on average; Table II). Thus the mid-anaphase delay likely represents surveillance mechanisms that effectively inactivate the microtubule assembly and motility mechanisms which normally produce further spindle elongation.
The dependency of the mid-anaphase delay on the RAD9 gene reveals that the DNA damage checkpoint continues to monitor chromosome integrity well after the start of anaphase, as well as before anaphase onset (Fig. 10). Thus RAD9-dependent DNA damage arrests the cell at any stage (G1, S, G2, and now mid-anaphase). This notion is similar to recent findings that checkpoint genes (RAD9, Siede et al., 1993; and the SAD1/RAD53 protein kinase, Allen et al., 1994) indeed control multiple checkpoints. Whether the damage leads to the same or different signal in each stage, or different signals in the same stage, is not well understood, but several genes are clearly involved at multiple stages of the cycle. Rad9p is constitutively expressed throughout the cell cycle (Schiestl et al., 1989), consistent with the proposal that this protein is involved in DNA surveillance throughout G1/S, G2/M, and anaphase/ cytokinesis. It is also likely that DNA lesions, in addition to a dicentric-induced aberration, are also subject to RAD9 surveillance at mid-anaphase. However, due to the rapidity of spindle elongation and the relatively extended lengths of G1/S and G2/M, the mid-anaphase delay would be difficult to identify in a population study. In this regard, we have examined the effect of a dominant negative form of topoisomerase I (top1-103), which also results in a RAD9-dependent cell cycle arrest (Levin et al., 1993). As reported previously, a large fraction of cells exhibited the large budded RAD9-dependent arrest morphology following 8 h of top1-103 induction. However, DAPI staining of the cells revealed two classes of arrest morphologies. About two-thirds of the cells displayed a single nucleus at the neck of large budded cells, while one-third exhibited elongated nuclei (sausage-shaped) spanning the neck region between the mother and bud (Beach, D., and K. Bloom, unpublished results). This second class of cells is identical in morphology to cells which arrest in midanaphase and indicates that a variety of DNA lesions can lead to delays in mid-anaphase.
The description of a mid to late-anaphase checkpoint leads to the notion that the major cyclin-dependent kinase–cyclin complexes, other regulatory kinases, e.g., DBF2 and 20 (Toyn and Johnston, 1994), cyclin destruction, or other anaphase-progression factors, are operative after anaphase onset (Irniger et al., 1995). In fact, H1 kinase levels remain elevated for prolonged periods in yeast cell populations following activation of the dicentric chromosome (Neff and Burke, 1992; Brock and Bloom, 1994). Cyclins and H1 kinase activity persist throughout anaphase in budding yeast, Drosophila, and nontransformed mammalian cells (Surana et al., 1993; Girard et al., 1995; Sigrist et al., 1995). In budding yeast, fully activated cyclins or overexpression of nondegradable forms of cyclin prevent spindle disassembly at telophase, rather than anaphase onset (Stueland et al., 1993; Surana et al., 1993), and high levels of cdc28/clb are found in cells arrested at telophase (cdc15; Surana et al., 1993; Irniger et al., 1995). The persistence of cyclin/clb complexes throughout anaphase raises the possibility that they may be involved in regulating the progression of anaphase directly, or by delaying anaphase progression in response to perturbation. The coexistence of subpopulations of degraded and protected cyclins (Irniger et al., 1995) is also suggestive of a role for the cdc28/cyclin B complex in mediating the progression of anaphase, spindle disassembly, and cytokinesis. Thus the sequential degradation of different cyclins may drive the progression from metaphase to telophase, or may be required to delay anaphase progression in the event of damage.
The description of a mid-anaphase transition raises two major questions. Upon sister chromatid separation, the cells lose a major pathway for repair of DNA damage. Therefore, why have a mid-anaphase checkpoint? Secondly, what perturbations are monitored by this checkpoint in the normal course of cell division? Several attributes of mitosis in budding yeast shed light on these issues. Yeast chromosomes have not been observed to align on a metaphase plate before anaphase onset. This has been thought to reflect the absence of chromosome condensation. However, Winey et al. (1995) demonstrated that kinetochore microtubules shorten asynchronously, reflecting the asynchronous movement of chromosomes to the poles. Mid-anaphase may therefore represent the last time in the cell cycle where errors in attachment may be rectified. Alternatively, the mid-anaphase transition could represent the last stage for DNA repair between sister chromatids. While FISH experiments (Guacci et al., 1994) and results from analysis of sister chromatids at the midanaphase delay (Fig. 6) indicate that sister centromeres have separated at this stage, the chromosome arms may not be completely disjoined. The uniformity of DAPI staining in cells with 4 µm spindles spanning the neck indicates that the bulk of the chromosomal DNA is evenly distributed within the nucleus. In contrast, the morphological transition to a bi-lobed nucleus results in substantial separation of chromosomal DNA. This most likely represents the final decatenation between sister chromatids and the irreversible commitment to chromosome separation (Fig. 10). In this view, the mid-anaphase transition represents the stage prior to the final commitment to complete DNA separation. The temporal coordination between completion of sister chromatid decatenation and force generation for spindle elongation necessary for preventing chromosome breakage may be the primary reason for the midanaphase pause, and one that may be important for all eukaryotic organisms. Loss of coordination of these events in transformed cells lacking mid-anaphase checkpoints may result in increased chromosome breakage, giving rise to chromosomal rearrangements, loss of genomic integrity, and hence, the neoplastic state.
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This work was supported by research grants from the National Institutes of Health General Medical Sciences (K. Bloom and E.D. Salmon).
Submitted: 6 June 1996
Revised: 15 November 1996
Please address all correspondence to Kerry Bloom, Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280. Tel.: 919962-1182; Fax: 919-962-1625; E-mail: ksb.fordham{at}mhs.unc.edu
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Allen JB, Zhou Z, Siede W, Friedberg EC & Elledge SJ. The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast, Genes Dev, 1994, 8, 2401–2415.
Brock JA & Bloom K. A chromosome breakage assay to monitor mitotic forces in budding yeast, J Cell Sci, 1994, 107, 891–902.[Abstract]
Copeland CS & Snyder M. Nuclear pore complex antigens delineate nuclear envelope dynamics in vegetative and conjugating Saccharomyces cerevisiae. , Yeast, 1993, 9, 235–249.[Medline]
Eshel D, Urrestarazu LA, Vissers S, Jauniaux JC, van Vliet-Reedijk JC, Planta RJ & Gibbons IR. Cytoplasmic dynein is required for normal nuclear segregation in yeast, Proc Natl Acad Sci USA, 1993, 90, 11172–11176.
Girard F, Fernandez A & Lamb N. Delayed cyclin A and B1 degradation in non-transformed mammalian cells, J Cell Sci, 1995, 108, 2599–2608.[Abstract]
Gorbsky GJ & Ricketts WA. Differential expression of a phosphoepitope at the kinetochores of moving chromosomes, J Cell Biol, 1993, 122, 1311–1321.
Guacci V, Hogan E & Koshland D. Chromosome condensation and sister chromatid pairing in budding yeast, J Cell Biol, 1994, 125, 517–530.
Hartwell LH & Weinert TA. Checkpoints: controls that ensure the order of cell cycle events, Science (Wash DC), 1989, 246, 629–634.
Hill A & Bloom K. Genetic manipulation of centromere function, Mol Cell Biol, 1987, 7, 2397–2405.
Hill A & Bloom K. Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae. , Mol Cell Biol, 1989, 9, 1368–1370.
Hoyt MA, Totis L & Roberts BT. S. cerevisiaegenes required for cell cycle arrest in response to loss of microtubule function, Cell, 1991, 66, 507–517.[Medline]
Irniger S, Piatti S, Michaelis C & Nasmyth K. Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast, Cell, 1995, 81, 269–278.[Medline]
Kahana JA, Schnapp BJ & Silver PA. Kinetics of spindle pole body separation in budding yeast, Proc Natl Acad Sci USA, 1995, 92, 9707–9711.
Koshland D, Rutledge L, Fitzgerald-Hayes M & Hartwell LH. A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae. , Cell, 1987, 48, 801–812.[Medline]
Levin NA, Bjornsti MA & Fink GR. A novel mutation in DNA topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest, Genetics, 1993, 133, 799–814.[Abstract]
Li R & Murray AW. Feedback control of mitosis in budding yeast, Cell, 1991, 66, 519–531.[Medline]
Li X & Nicklas RB. Mitotic forces control a cell-cycle checkpoint, Nature (Lond), 1995, 373, 630–632.[Medline]
Li YY, Yeh E, Hays T & Bloom K. Disruption of mitotic spindle orientation in a yeast dynein mutant, Proc Natl Acad Sci USA, 1993, 90, 10096–10100.
Lydall D & Weinert T. Yeast checkpoint genes in DNA damage processing: implications for repair and arrest, Science (Wash DC), 1995, 270, 1488–1491.
Murray A. a. Cyclin ubiquitination: the destructive end of mitosis, Cell, 1995, 81, 149–152.[Medline]
Murray AW. b. The genetics of cell cycle checkpoints, Curr Opin Genet Dev, 1995, 5, 5–11.[Medline]
Nasmyth K. Control of the yeast cell cycle by the Cdc28 protein kinase, Curr Opin Cell Biol, 1993, 5, 166–179.[Medline]
Neff MW & Burke DJ. A delay in the Saccharomyces cerevisiaecell cycle that is induced by a dicentric chromosome and dependent upon mitotic checkpoints, Mol Cell Biol, 1992, 12, 3857–3864.
Pringle JR, Preston RA, Adams AEM, Stearns T, Drubin DG, Haarer BK & Jones EW. Fluorescence microscopy methods for yeast, Methods Cell Biol, 1989, 31, 358–435.
Salmon ED, Inoue T, Desai A & Murray AW. High resolution multimode digital imaging system for mitosis studies in vivo and in vitro. , Biol Bull (Woods Hole), 1994, 187, 231–232.[Medline]
Saunders WS & Hoyt MA. Kinesin-related proteins required for structural integrity of the mitotic spindle, Cell, 1992, 70, 451–458.[Medline]
Saunders WS, Koshland D, Eshel D, Gibbons IR & Hoyt MA. Saccharomyces cerevisiaekinesin- and dynein-related proteins required for anaphase chromosome segregation, J Cell Biol, 1995, 128, 617–624.
Schiestl RH, Reynolds P, Prakash S & Prakash L. Cloning and sequence analysis of the Saccharomyces cerevisiaeRAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage, Mol Cell Biol, 1989, 9, 1882–1896.
Siede W, Friedberg AS & Friedberg EC. RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae. , Proc Natl Acad Sci USA, 1993, 90, 7985–7989.
Sigrist S, Jacobs H, Stratmann R & Lehner CF. Exit from mitosis is regulated by Drosophilafizzy and the sequential destruction of cyclins A, B, and B3, EMBO (Eur Mol Biol Organ) J, 1995, 14, 4827–4838.[Medline]
Skibbens RV, Skeen VP & Salmon ED. Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push–pull mechanism, J Cell Biol, 1993, 122, 859–875.
Spencer F & Hieter P. Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae. , Proc Natl Acad Sci USA, 1992, 89, 8908–8912.
Stueland CS, Lew DJ, Cismowski MJ & Reed SI. Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae. , Mol Cell Biol, 1993, 13, 3744–3755.
Surana U, Amon A, Dowzer C, McGrew J, Byers B & Nasmyth K. Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast, EMBO (Eur Mol Biol Organ) J, 1993, 12, 1969–1978.[Medline]
Toyn JH & Johnston LH. The Dbf2 and Dbf20 protein kinases of budding yeast are activated after the metaphase to anaphase cell cycle transition, EMBO (Eur Mol Biol Organ) J, 1994, 13, 1103–1113.[Medline]
Wang Y & Burke DJ. Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. , Mol Cell Biol, 1995, 115, 6838–6844.
Weinert TA & Hartwell LH. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. , Science (Wash DC), 1988, 241, 317–322.
Weinert T & Lydall D. Cell cycle checkpoints, genetic instability and cancer, Semin Cancer Biol, 1993, 4, 129–140.[Medline]
Weinert TA, Kiser GL & Hartwell LH. Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair, Genes Dev, 1994, 8, 652–665.
Wells WA. The spindle assembly checkpoint: aiming for a perfect mitosis, every time, Trends Cell Biol, 1996, 6, 228–234.[Medline]
Winey M, Mamay CL, O'Toole ET, Mastronarde DN, Giddings TH Jr, McDonald KL & McIntosh JR. Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiaemitotic spindle, J Cell Biol, 1995, 129, 1601–1615.
Yeh E, Skibbens RV, Cheng JW, Salmon ED & Bloom K. Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae. , J Cell Biol, 1995, 130, 687–700.
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