53BP1 is a vertebrate BRCT motif protein, originally described as a direct interactor of p53, which has recently been shown to be implicated in the early response to DNA damage. Upon DNA damage, 53BP1 re-localises to discrete nuclear foci that are thought to represent sites of DNA lesions and becomes hyperphosphorylated. Several observations suggest that 53BP1 is a direct substrate for the ataxia telangiectasia mutated (ATM) kinase. So far, 53BP1 behaviour during mitosis has not been reported in detail. We have examined 53BP1 subcellular distribution in mitotic cells using several antibodies against 53BP1, and ectopic expression of GFP-tagged 53BP1. We found that 53BP1 significantly colocalised with CENP-E to kinetochores. 53BP1 is loaded to kinetochores in prophase, before CENP-E, and is released by mid-anaphase. By expressing various GFP-tagged 53BP1 truncations, the kinetochore binding domain has been mapped to a 380 residue portion of the protein that excludes the nuclear localisation signal and the BRCT motifs. Like many kinetochore-associated proteins involved in mitotic checkpoint signalling, more 53BP1 appears to accumulate on the kinetochores of chromosomes not aligned on the metaphase plate. Finally, we show that 53BP1 is hyperphosphorylated in mitotic cells, and undergoes an even higher level of phosphorylation in response to spindle disruption with colcemid. Our data suggest that 53BP1 may have a role in checkpoint signalling during mitosis and provide the evidence that DNA damage response machinery and mitotic checkpoint may share common molecular components.
Vertebrate kinetochores are large proteinaceous structures that assemble on centromeres and link mitotic chromosomes to the microtubules during mitosis ( Craig et al., 1999; Maney et al., 2000). In addition to this structural role, it is thought that kinetochores govern the mechanical movements of chromosomes through the action on the spindle microtubules of several kinetochore-bound molecular motors such as CENP-E ( Schaar et al., 1997), the mitotic centromere-associated kinesin (MCAK) ( Maney et al., 1998), and the dynein-dynactin complex ( Echeverri et al., 1996). One of the most documented functions of the kinetochore remains its pivotal role in the mitotic checkpoint, a mechanism by which cells are able to delay anaphase onset, by inhibiting the anaphase promoting complex (APC), until all the chromosomes have achieved a proper bipolar attachment to the spindle ( Wassmann and Benezra, 2001). Vertebrate kinetochores contain the molecular machinery for the mitotic checkpoint. Many kinetochore-associated proteins including MAD1 ( Chen et al., 1998), MAD2 ( Li and Benezra, 1996), BUB1 ( Taylor and McKeon, 1997), BUBR1 ( Chan et al., 1999), BUB3 ( Kalitsis et al., 2000), activated MAP kinase ( Wang et al., 1997), the kinesin like protein CENP-E ( Abrieu et al., 2000), hROD and hZW10 ( Chan et al., 2000) have been shown to be essential for the proper maintenance of the mitotic checkpoint.
A major function of kinetochores, which is linked to their role in the mitotic checkpoint, is to sense the attachment of the spindle microtubules and the tension subsequently generated by the bipolar attachment. This is reflected by the fact that in response to microtubule attachment, the biochemical composition of kinetochore is dramatically modified. First, some of the kinetochore signalling proteins are released in response to the capture of the kinetochore by the spindle. These are present at higher levels on kinetochores of chromosomes that are not aligned properly on the metaphase plate ( Chen et al., 1996; Jablonski et al., 1998; Kallio et al., 1998; Martinez-Exposito et al., 1999; Waters et al., 1998; Zecevic et al., 1998). Second, the phosphorylation state of some kinetochore components is dependent on the attachment and/or the tension states, a notion supported, for example, by the fact that the phosphoepitope 3F3/2 is only detected on the kinetochores that are not under tension ( Gorbsky and Ricketts, 1993).
53BP1 (p53 binding protein 1) is a vertebrate BRCT protein that was originally identified in a yeast two-hybrid screen as a protein that interacts with the p53 tumour suppresser ( Iwabuchi et al., 1994). The fact that 53BP1 stimulates p53-mediated transcription activation in transfection assays has provided some insights into the functional relationships between both proteins ( Datta et al., 1996; Iwabuchi et al., 1998). The C-terminus of 53BP1 contains a tandem repeat of BRCT (BRCA1 C-terminus) motifs. This motif has been found in a large number of proteins involved in various aspects of the cell-cycle control and the DNA damage response, and is thought to mediate protein-protein interactions ( Bork et al., 1997). Examination of 53BP1 behaviour upon activation of the cellular response to DNA damage provided several lines of evidence clearly implicating 53BP1 in the DNA damage response. First, similarly to some proteins known to have a role in DNA damage repair and checkpoint signalling, including BRCA1, Rad51 and the Rad50/Mre11/NBS1 complex ( Petrini, 1999), 53BP1 redistributes to multiple discrete nuclear foci within minutes after exposure of cells to ionising irradiation, whereas in normal interphase cells it is diffusely distributed over the chromatin ( Anderson et al., 2001; Rappold et al., 2001; Schultz et al., 2000). The 53BP1 foci co-localise with those of the Mre11/Nbs1/Rad50 complex ( Anderson et al., 2001; Schultz et al., 2000) and phosphorylated γH2AX ( Rappold et al., 2001; Schultz et al., 2000). The latter has been demonstrated to coincide with the sites of double-strand DNA breaks ( Nelms et al., 1998; Rogakou et al., 1999). Not only is 53BP1 spatially associated with phosphorylated γH2AX foci, both proteins have recently been shown to co-immunoprecipitate if the cells are treated with ionising irradiation ( Rappold et al., 2001). Another behaviour of 53BP1 shared with some DNA damage response signaling factors is that it becomes hyper-phosphorylated upon DNA damage, several observations suggesting that 53BP1 is a direct substrate of the ATM (ataxia telangiectasia mutated) kinase after ionising radiation treatment ( Anderson et al., 2001; Rappold et al., 2001; Schultz et al., 2000).
Although significant progress towards 53BP1 function has been recently made, all studies on 53BP1 performed so far have focused on interphase cells. 53BP1 behaviour during mitosis has never been described in detail. Here, using several antibodies against human and mouse 53BP1, we have examined 53BP1 subcellular distribution in mitotic cells. Surprisingly, we found that 53BP1 localises to kinetochores and is hyper-phosphorylated during mitosis under conditions where the spindle checkpoint is activated, suggesting that, in addition to its role in DNA damage response, 53BP1 has a role in signalling at the kinetochore during mitosis.
Materials and methods
Mouse 53BP1 cDNA cloning
A 3.1 kb mouse EST (expressed sequence tag), from the UK HGMP resource centre, coding for the C-terminal region of the mouse orthologue of 53BP1 was used to screen a mouse brain cDNA library (Stratagene). After several rounds of screening, the most 5′ sequence was obtained. A 6579 bp cDNA was assembled encoding a 1957-residue protein referred as to m53BP1 sharing 80% overall identity with the human 53BP1 protein. Probes were labelled with 32P using the High Prime random priming kit (Roche). Plaque hybridisation was performed as described ( Jullien et al., 1997). The m53BP1 sequences are available from the DDBJ/EMBL/GenBank databases under accession number AJ414734.
Cell culture and synchronisation
HeLa cells and NIH3T3 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 5 μg/ml gentamycin, in a 5% CO2 humidified 37°C incubator. Mitotic HeLa cells were obtained by a double-thymidine block: 15 hours of 2 mM thymidine block followed by a 9 hour release. Microtubule-depolymerising drug colcemid was added to 0.1 μg/ml, 8 hours after the second release from the thymidine block. After a 2 hour incubation with colcemid, mitotic cells were harvested from culture flasks by mechanical shake off.
To raise antibodies against the N-terminal region and C-terminal domain of the mouse 53BP1 (m53BP1), cDNA sequence encoding the first 355 residues (N-m53BP1) and amino acid position 1293 to 1809 (C-m53BP1) of m53BP1 were amplified by PCR from the cloned m53BP1 cDNA and cloned into pRSETb (Invitrogen). Both histidine-tagged proteins were expressed in E. coli BL21(DE3) harbouring pLysS. N-m53BP1 was purified on Ni-agarose resin (Qiagen), run on a SDS polyacrylamide gel, eluted by diffusion and used as an antigen to immunise rabbits. C-m53BP1 antigen purification and affinity-purification of antibodies were performed as previously described ( Anderson et al., 2001).
All the EGFP:m53BP1 fusion constructs used in transient transfection were made in the pEGFP-C1 (Clontech). To express EGFP fused to m53BP1 protein, m53BP1 full length ORF was cloned at the SacII-Apa1 sites of pEGFP-C1 by introduction of a SacII site before the initiation codon by PCR. Constructs expressing EGFP:m53BP1753-1957, EGFP:m53BP11294-1957, EGFP:m53BP11-1601, EGFP:m53BP11-1139, EGFP:m53BP1753-1601 and EGFP:m53BP11294-1601 were obtained by subcloning the m53BP1 cDNA XhoI-ApaI, HindIII-ApaI, SacII-EcoRV, SacII-BamHI, Xho-EcoRV, HindII-EcoRV fragments, respectively, into pEGFP-C1. m53BP11140-1703 coding sequence was generated by PCR introducing an EcoRI site at the 5′ end and a BamHI site at the 3′ end, and was cloned into pEGFP-C1 digested with EcoRI-BamHI. m53BP11140-1601 coding sequence was produced by digesting m53BP11140-1703 PCR product with EcoRI and EcoRV and cloned into pEGFP-C1 digested EcoR1-SmaI. m53BP11169-1601 and m53BP11220-1601 were generated by PCR using appropriate 5′ oligos in combination with the 3′ oligo used for m53BP11140-1703 and cloned after being digested EcoRI-EcoRV into pEGFP-C1 EcoRI-SmaI. m53BP11220-1515 coding sequence was generated by PCR using the 5′ oligo used for m53BP11140-1703 and a specific 3′ oligo, and cloned EcoRI-BamHI in pEGFP-C1. All the constructs were checked by sequencing.
Immunofluorescence microscopy and transfections
Cells grown on poly-L lysine coated glass coverslips were synchronized by a double thymidine block. Cells were transiently transfected by calcium phosphate precipitation. Briefly, 5 μg of DNA was precipitated and applied onto a 22×22 mm coverslip for 12 hours. Coverslips were then washed with PBS and the double-thymidine block was performed. For immunofluorescence staining, 9 hours after release from the second thymidine block, cells were briefly rinsed with PBS, fixed with methanol at –20°C for 20 minutes, and rehydrated with PBS three times for 5 minutes. Blocking and incubation with the antibodies were essentially performed as described previously ( Anderson et al., 2001). Affinity purified anti-human or mouse 53BP1 antibodies were used at a concentration of 10 μg/ml, anti-CENP-B monoclonal antibody, ACA human autoimmune antibodies and anti-CENP-E monoclonal antibody (mAb177; a gift from T. J. Yen, Fox Chase Cancer Center, Philadelphia, PA), were used at a dilution 1/20, 1/1000, and 1/500, respectively. As negative control, preimmune sera were used at a 1/1000 dilution. Secondary antibodies anti-Rabbit IgG-FITC (Sigma), anti-mouse IgG-Texas Red (Vector) and anti-human IgG-FITC (Vector) were used at a 1/200 dilution. DNA was stained by the use of the Vectashield® mounting medium containing DAPI (Vector). Images were recorded using either a Zeiss Axioskop microscope equipped with a VYSIS image recording system ( Fig. 3; Fig. 4) or a Delta Vision microscope (Applied Precision) based on an Olympus IX-70 inverted microscope with a cooled couple-charged device camera (CH350L, Photometrics) ( Fig. 2; Fig. 5; Fig. 6B). Three-dimensional data sets of selected cells were collected with the Delta Vision, deconvolved, projected onto a single plane and exported as TIFF files to be processed using Adobe PhotoShop. The intensities of 53BP1 kinetochore signals were quantified by Data Inspector tool of the Delta Vision microscope ( Adams et al., 2001). We measured the integrated intensity of a kinetochore at the appropriate wavelengths for detection of 53BP1 and ACA. The signal strength of 53BP1 was defined as a ratio of the 53BP1 intensity to that of ACA which has shown to be constant throughout the cell cycle ( Earnshaw et al., 1986). At least 10 kinetochores were examined and the values were averaged.
Immunoblotting and immunoprecipitation
Protein extract preparation from asynchronous or mitotic cells, estimation of the protein concentration, immunoprecipitations and western blotting were performed as described previously ( Anderson et al., 2001).
53BP1 localises to the kinetochore
In order to investigate 53BP1 subcellular distribution during mitosis, two polyclonal antibodies, were independently raised against the N-terminal (N-m53BP1) or C-terminal (C-m53BP1) region of mouse 53BP1 (m53BP1, see Materials and Methods). The specificity of the affinity-purified antibodies against the N-m53BP1 and C-m53BP1 was examined by western blot on mouse cell protein extracts ( Fig. 1); they both gave a major single band signal ( Fig. 1, lanes 2,4) of ∼300 kDa ( Anderson et al., 2001). The pre-immune sera from the same rabbits gave no or very little signal ( Fig. 1, lanes 1,3). In addition, we used previously described antibodies directed against human 53BP1 (referred to here as 53BP1) ( Anderson et al., 2001).
The antibodies were used to immunostain HeLa and NIH3T3 cells in combination with anti-centromeric antibodies (ACA), which recognise CENP-A, -B and -C ( Earnshaw et al., 1986). When mitotic cells in pro-metaphase were observed, 53BP1 ( Fig. 2a-d) and m53BP1 (stained with the anti-N-m53BP1; Fig. 2e-h) were detected as foci on the condensed chromosomes in addition to a diffuse cytoplasmic signal ( Fig. 2c,g; Fig. 4). Preimmune sera gave a very low background signal (data not shown). Merging the 53BP1 or the m53BP1 images with the ACA signal revealed that 53BP1 foci and centromeres were adjacent although not exactly coincident ( Fig. 2b,f). When centromeres of individual chromosomes were observed, 53BP1 paired foci appeared external to the ACA doublet ( Fig. 2, see magnified area in b,f; 53BP1 in red; ACA in green). The fact that 53BP1 appeared to bracket the centromeres was even more obvious in prometaphase cells in which 53BP1 localisation was compared with the centromeric protein CENP-B ( Cooke et al., 1990) ( Fig. 2i-l, see magnified area in j), suggesting that 53BP1 was localised to the kinetochores. An identical staining pattern was obtained with the antibodies against C-m53BP1 (data not shown). Under the same experimental conditions, none of the three related preimmune sera gave any signal associated with centromeric staining (data not shown).
To address the relationship between 53BP1 mitotic foci and kinetochores in more detail, double immuno-staining was performed with an antibody directed against CENP-E, a component of the kinetochore corona ( Cooke et al., 1997; Yao et al., 1997). When the 53BP1 image was merged with CENP-E, partial overlaps were observed between the two signals ( Fig. 2, m-p; Fig. 6B; note that the overlaps of red and green signals yield yellow signal in merged images). In some examples, 53BP1 appeared to extend beyond the boundary of the CENP-E staining ( Fig. 2n, see magnified area). Thus three independently prepared antibodies against human and mouse 53BP1 localised the protein to chromosomal structures that were spatially linked but not exactly coincident with centromeric antigens (e.g. CENP-B), but significantly co-localised with the outer kinetochore protein CENP-E.
Identification of the 53BP1 kinetochore binding domain
To further confirm the kinetochore association of 53BP1, green fluorescent protein (GFP) fused to full length m53BP1 was expressed transiently in HeLa cells and its distribution was examined in mitotic cells. The kinetochore marker CENP-E was detected by immunostaining as a control. The GFP-m53BP1 fusion protein was found to temporally (data not shown) and spatially localise to kinetochores in mitotic cells ( Fig. 3Ba). This excluded the unlikely possibility that the detection of 53BP1 at kinetochores was an artefact of the indirect immunofluorescence protocol.
Next we attempted to identify the sub-domain of 53BP1 that was sufficient to localise the GFP fusion to the kinetochore. Various portions of the protein were fused at their N-terminus to GFP, and a total of 11 different truncations were analysed ( Fig. 3A). Two overlapping deletions, GFP:m53BP1753-1957 and GFP:m53BP11-1601 targeted to the kinetochores ( Fig. 3b,d). These initial results revealed that the region between amino acids 753 and 1601 contained a kinetochore-binding domain. In agreement with this, transiently expressed GFP:m53BP1753-1601 was found to associate with the kinetochores ( Fig. 3Bf). The larger N-terminal (GFP:m53BP11294-1957) and C-terminal (GFP:m53BP11-1139) deletion constructs both failed to associate with kinetochores ( Fig. 3Bc,e). We noted that the former construct, associated with the mitotic chromatin ( Fig. 3Bc) giving a GFP signal chromosome shaped without any detectable focal signal. To further delineate the N-terminal boundary of the targeting domain, we assayed for the kinetochore localisation of four increasing N-terminal deletions of the 753-1601 region. GFP:m53BP11140-1601, GFP:m53BP11169-1601 and GFP:m53BP11220-1601 were all kinetochore associated, whereas a further deletion to the amino acid 1294 (GFP:m53BP11294-1601) abolished the kinetochore targeting ( Fig. 3Bh-k). We then deleted the 1220-1601 region from the C-terminus. When a construct expressing GFP:m53BP11220-1515 was transiently transfected in the cells, again no detectable GFP signal was seen at the kinetochores ( Fig. 3Bl) suggesting that the C-terminal limit of the kinetochore binding domain was located between amino acids 1515 and 1601.
This GFP-53BP1 domain analysis also allowed us to map the nuclear localisation signal. All C-terminal deletion constructs terminating at or before amino acid 1601 were excluded from the nucleus during interphase ( Fig. 3A, N.L.). Among those nuclear-excluded mutants was GFP:m53BP11140-1601. Interestingly, the same construct with a C-terminal extension of 102 amino acid residues, namely GFP:m53BP11140-1703, properly accumulated in the interphase nucleus ( Fig. 3A). These data showed that 53BP1 nuclear localisation signal was located between amino acids 1601 and 1703, a domain not required for 53BP1 kinetochore recruitment.
Taken together, the minimal m53BP1 kinetochore binding domain resides between residues 1220 and 1601, a domain that overlaps neither with the nuclear localisation signal, nor with the C-terminal BRCT motif tandem.
Time window of 53BP1 association with kinetochores
We next examined in details at which stages of mitosis 53BP1 was associated with the kinetochores by co-staining HeLa cells for 53BP1 and CENP-B, which is constitutively bound to centromeres. In interphase cells, 53BP1 has been shown to associate to a few bright foci in some cells, whereas upon DNA damage, 53BP1 redistributes to multiple nuclear foci in all the cells ( Anderson et al., 2001). Therefore, we first addressed whether 53BP1 interphase foci would exhibit any spatial relationship with centromeres detected with CENP-B. As shown in Fig. 4a, no significant co-localisation was observed between the two families of foci, indicating that 53BP1 was not associated with centromeres during interphase. The earliest stage of mitosis at which 53BP1 dots were seen to be spatially linked to CENP-B signal was prophase, as judged from the chromatin morphology ( Fig. 4b, compare DAPI staining with a). To further narrow the time-frame when 53BP1 assembled onto kinetochores, we compared the timing of association of 53BP1 to that of CENP-E, which is loaded by early pro-metaphase ( Chan et al., 1998). We double-stained HeLa cells with anti-53BP1 and anti-CENP-E antibodies ( Fig. 4c,d). In cells exhibiting a chromatin morphology characteristic of prophase (compare DAPI staining in b and c), although 53BP1 paired foci were visible, we consistently failed to detect coincident CENP-E signal ( Fig. 4c). At a later stage, in early pro-metaphase cells, a bright 53BP1 signal was reproducibly associated with CENP-E dots ( Fig. 4d). These results suggested that 53BP1 was assembled onto kinetochores in prophase before the recruitment of CENP-E.
53BP1 kinetochore-associated signal peaked in late prophase to early prometaphase ( Fig. 4d) and then gradually diminished. Interestingly, during prometaphase, chromosomes that were not aligned to the metaphase plate always exhibited a much stronger 53BP1 signal compared with aligned chromosomes ( Fig. 5c). The difference was approximately fourfold from measuring the signal intensities of individual kinetochores (1.61±0.38 versus 0.38±0.12; for the quantification procedure see Materials and Methods). At metaphase, 53BP1 was still detected at kinetochores of the aligned chromosomes, however, the level was significantly lower (0.27±0.15; Fig. 4e). A weak signal was observed in early anaphase when sister chromatids had separated ( Fig. 4f). By mid-anaphase, 53BP1 was no longer detectable at the kinetochores ( Fig. 4g) and from this stage to late anaphase the protein appeared excluded from the condensed chromosomes. 53BP1 started to associate again with chromatin by telophase ( Fig. 4h), presumably when nuclear envelope reassembled. During all stages of mitosis when 53BP1 was associated to the kinetochores, prominent 53BP1 staining was also detected in the cytoplasm indicating that only a fraction of the total 53BP1 pool was bound to kinetochores ( Fig. 4, 53BP1 panels).
53BP1 is hyperphosphorylated in mitotic cells
53BP1 was immunoprecipitated from HeLa cell extracts prepared from non-synchronised, mitotic, and mitotically blocked cells with colcemid, a microtubule depolymerising drug, and its electrophoretic mobility was examined by western blotting ( Fig. 6A). 53BP1 mobility was clearly reduced in mitotic cells relative to unsynchronized cells ( Fig. 6A, lane 1 versus 2). In cells blocked with colcemid, 53BP1 exhibited an even slower mobility compared with normal mitotic cells ( Fig. 6A, lane 4 versus 2). These were due to phosphorylation as the slower mobility of 53BP1 was diminished after protein phosphatase treatment (lanes 3,5). These results showed that, in mitosis, 53BP1 is hyperphosphorylated to two different levels, most of the 53BP1 pool being subjected to the highest level of hyperphosphorylation after disruption of the spindle.
In cells treated with colcemid, 53BP1 ( Fig. 6Bb,c), like CENP-E ( Fig. 6Bb,d), exhibited a strong crescent-shaped signal on the kinetochores (compare with the cells in natural mitosis without colcemid treatment in Fig. 2m-p). We noticed that the kinetochore signal of 53BP1 was significantly stronger (approximately four times) in cells treated with colcemid in comparison with those in normal mitotic cells (1.24±0.39 versus 0.27±0.15). Taken along with the hyper-phosphorylation of the protein in the presence of the microtubule de-polymerising drug, these data suggest that 53BP1 is responsive to the lack of spindle microtubules. In addition, the data demonstrated that the association of 53BP1 to kinetochores did not require spindle microtubules.
We have previously reported that, during interphase, several characteristics of 53BP1 behaviour upon DNA damage classified this protein as a molecular component of the early cellular response to genotoxic stress ( Anderson et al., 2001). Here, we have examined the localisation of 53BP1 in mitotic cells and showed that 53BP1 is a kinetochore-associated protein that exhibits behaviour characteristic of proteins involved in the metaphase checkpoint.
53BP1 kinetochore localisation was directly demonstrated by two lines of evidence. First, three antibodies independently raised against human and mouse 53BP1 orthologues specifically detected 53BP1 at kinetochores. Second, transiently expressed GFP-tagged 53BP1 properly localised to kinetochores ( Fig. 3). We have shown that 53BP1 is bound to the kinetochore, but not to the underlying centromere, by comparing its localisation with those of the centromeric DNA-binding protein CENP-B ( Cooke et al., 1990) and CENP-E, a microtubule-motor protein that resides within the fibrous corona of kinetochore ( Cooke et al., 1997; Yao et al., 1997). We have never observed any co-localisation between CENP-B and 53BP1, whereas 53BP1 and CENP-E were found significantly coincident, although the overlap was not always complete ( Fig. 2). The spatial overlap between 53BP1 and CENP-E suggests that 53BP1 is a component of the kinetochore corona.
In interphase cells, we did not detect any co-localisation of 53BP1 foci and centromeres indicating that 53BP1 is associated with kinetochores only during mitosis. So far, 53BP1 is the only DNA damage repair/checkpoint protein localised to the mitotic kinetochores. It is unlikely that the kinetochore localisation of 53BP1 is due to DNA lesions on the centromere DNA particularly since the region of the kinetochore to which 53BP1 localises does not contain detectable DNA ( Cooke et al., 1990).
Using GFP-tagged 53BP1 truncations, the kinetochore targeting domain has been localised to a 380 residue region located in the C-terminal half of 53BP1 ( Fig. 3A). This domain does not contain the C-terminal BRCT motifs excluding p53 from this interaction since 53BP1 has been shown to interact with p53 through its BRCT motifs ( Iwabuchi et al., 1994).
One characteristic of the kinetochores is the dynamic association and dissociation of its molecular components during mitosis. 53BP1 appears to associate with kinetochore at prophase before CENP-E and shows a significantly higher level of association with chromosomes not aligned at the metaphaphase plate. A number of vertebrate kinetochore proteins involved in checkpoint signalling including BUB1, BUBR1, CENP-E, BUB3, Mad2 and p55CDC ( Chan et al., 1998; Jablonski et al., 1998; Kallio et al., 1998; Martinez-Exposito et al., 1999; Waters et al., 1998) show similar behaviour. Moreover, in cells mitotically blocked with a microtubule depolymerising drug, in which all the kinetochores are unattached, a strong crescent shaped 53BP1 signal was observed at the surface of the kinetochores ( Fig. 5). A reduced level of 53BP1 was detected at metaphase kinetochores by which stage the kinetochore association of the previously characterised checkpoint components is significantly decreased. 53BP1 is completely released from the kinetochore later, after the metaphase-anaphase transition.
The kinetochore is directly involved in spindle checkpoint signalling function. In the absence of proper attatchment to the mitotic spindle, kinetochore-associated kinases are activated, leading to subsequent phosphorylation of signalling components such as Mad1p ( Hardwick and Murray, 1995). We have observed that in cells blocked in mitosis with colcemid, 53BP1 undergoes a higher level of hyper-phosphorylation compared with the level detected in normal mitotic cells ( Fig. 6). This suggests that, like Mad1p, 53BP1 is a substrate of kinase(s) activated when the spindle checkpoint is challenged. As kinetochores are known to contain cdc2 ( Rattner et al., 1990), MAP kinase ( Zecevic et al., 1998), BUB1 ( Taylor and McKeon, 1997), and BUBR1 ( Chan et al., 1998), any one of these kinases may be involved in 53BP1 hyperphosphorylation in mitotically blocked cells. In vertebrate cells, the kinetochore-associated spindle-checkpoint kinase BUBR1 has been shown to be similarly hyperphosphorylated upon depletion of the spindle microtubules ( Chan et al., 1999).
Our results suggest that 53BP1, a component of the DNA damage response may also play a role during mitosis in checkpoint signalling at the kinetochore. Other studies have also suggested that proteins may be shared by the DNA damage response machinery and other cell cycle checkpoints. For example, mouse embryonic fibroblast cells deficient for p53, or for BRCA1, show abnormal centrosome amplification as well as defects in their DNA damage response ( Fukasawa et al., 1996; Xu et al., 1999). There might also be a link between the spindle checkpoint and G1/S transition control. It has been shown that cells do not enter S phase after escaping a prolonged spindle-checkpoint arrest (a process called adaptation), and p53 and possibly the ATM kinase are involved in this suppression of re-entering S phase without chromosome separation ( Lanni and Jacks, 1998; Shigeta et al., 1999). Our studies suggest that 53BP1 may be another protein with a common role in damage and mitotic checkpoints. These results are beginning to provide evidence for a common link between what were previously thought to be mechanically unrelated cell cycle checkpoints.
We are grateful to Catherine Henderson and Alan Mcnee for antibody preparation, T. J. Yen for the anti-CENP-E antibody and Kevin Hardwick, Hiro Ohkura and Takashi Toda for comments on the manuscript. This work was supported by the Wellcome Trust.
- Accepted September 19, 2001.
- © The Company of Biologists Limited 2002