It is essential that the cell does not segregate its chromosomes until after DNA replication and spindle assembly have been completed. The DNA damage, DNA replication and spindle assembly checkpoint pathways prevent passage through the metaphase/anaphase transition when DNA is damaged or when DNA replication or spindle assembly respectively are incomplete.
Figure 1.2 Regulation o f the Metaphase/Anaphase transition. Sister chromatid
segregation requires destruction of the anaphase inhibitor Pdsl which binds to and inhibits
the E sp l protease. P d sl is destroyed by APC/C"^^^^-dependent ubiquitin-mediated
proteolysis, liberating E sp l. Espl-dependent cleavage of Sccl leads to loss of sister
chromatid cohesion and triggers sister chromatid separation. Phosphorylation of Sccl by
Cdc5 promotes its cleavage, and Cdc5 may also activate the metaphase/anaphase transition
by phosphorylating and activating APC/C^^^^°. The DNA damage, DNA replication, and
spindle assembly checkpoint pathways arrest cells in metaphase. Activation of the DNA
damage checkpoint leads to inhibition of Cdc5, and phosphorylation of P dsl, which renders
it resistant to APC/C^'^'^^. In contrast, the spindle assembly checkpoint is though to stabilise
DNA damage
Spindle assembly
checkpoint
checkpoint
Cdc20
Cdc5
APC/C
P d sl
Esp1
Esp1
S o d
Metaphase
Anaphase
Sister chromatid
separation
1.7.2.3.1 The DNA checkpoint pathways
The DNA damage and replication checkpoint pathways are extremely complex. DNA damage generated by ultraviolet or ionising radiation, or stalled DNA replication (for example by addition of hydroxyurea) are sensed by different proteins but the signals appear to converge at the M ecl kinase (reviewed by Lowndes and Murguia, 2000). In response to DNA damage, M ecl activates a number of signalling cascades, including a Rad53- dependent pathway which inhibits Cdc5 (Sanchez et al., 1999) and consequently exerts a negative influence on the metaphase/anaphase transition. M ecl also activates the Chkl kinase, which phosphorylates Pdsl, rendering it resistant to APC/C-mediated proteolysis (Lowndes and Murguia, 2000; Sanchez et at., 1999). Activation of the DNA replication (S- phase) checkpoint pathway also leads to stabilisation of Pdsl, but the mechanism is less well defined (Clarke and Gimenez-Abian, 2000). Thus DNA damage or incomplete DNA replication both result in a checkpoint arrest in metaphase, giving the cell time to correct the fault (Figure 1.2). It is worth noting that cells do not arrest indefinitely following checkpoint activation, since if the cell is unable to correct the problem it is better to attempt mitosis than to die. This phenomenon is termed adaptation, and interestingly, in budding yeast, Cdc5 is involved in adaptation to the DNA damage response, perhaps by overcoming its Rad53-dependent inhibition (Toczyski et al., 1997).
1.7.2.3.2 The spindle assembly checkpoint (SAC)
The spindle assembly checkpoint (SAC) arrests the cell cycle in metaphase in response to defects in the mitotic spindle (reviewed by Skibbens and Hieter, 1998). These include defects in spindle pole body duplication, kinetochore function, microtubule polymerisation and microtubule motor proteins (Hardwick et al., 1999). Experimentally, the checkpoint is usually activated by microtubule depolymerising drugs such as nocodazole or benomyl (Skibbens and Hieter, 1998).
The components of the budding yeast SAC were identified by screening for mutants which fail to arrest the cell cycle in the presence of microtubule depolymerising drugs (Hoyt et al..
1991; Li and Murray, 1991). These screens identified six genes necessary for SAC function, M ADl-3 and BUB 1-3, although it has since been shown that BUB2 forms a separate branch of the SAC (see 1.8.2.3). The M PSl protein kinase, which functions in SPB duplication (1.6.2.2) is also required for spindle checkpoint function, since unlike other SPB duplication mutants, m psl-1 cells do not display a SAC arrest (Weiss and Winey, 1996). The MAD and BUB genes are not essential for viability in budding yeast, although cells exhibit elevated levels of chromosome loss if the checkpoint genes are deleted, suggesting that the checkpoint is important for high fidelity chromosome segregation in an unperturbed cell cycle. In higher eukaryotes, the checkpoint components are essential genes, suggesting that the checkpoint has evolved into a fundamental element of cell cycle control (reviewed by Skibbens and Hieter, 1998).
It is not clear how the SAC components sense spindle abnormalities. Experiments in vertebrate cells suggest that the checkpoint responds to a failure to establish either kinetochore-microtubule attachment, or microtubule tension (Skibbens and Hieter, 1998). These two possibilities are not exclusive, and the cell may sense both attachment and tension (Skoufias et al., 2001). Consistent with both models, components of the SAC localise to kinetochores (Skibbens and Hieter, 1998). Moreover, the Xenopus Mad2 homologue localises predominantly to kinetochores with fewer microtubules attached (Chen et al., 1996), suggesting that Mad2 senses attachment of spindle microtubules to kinetochore binding sites.
Although the order of function of the SAC has not been established, the M psl and B ubl kinases are thought to function together upstream of the other SAC components, since over expression of M psl, or a dominant mutant form of B ubl, induces a metaphase arrest, which is dependent upon the remaining SAC components (including Bubl and M psl respectively) (Farr and Hoyt, 1998; Hardwick et a l, 1996). M psl phosphorylates M adl in vitro and in vivo (Hardwick et a l, 1996), whilst Bubl phosphorylates Bub3 in vitro, suggesting that Bub3 may be a Bubl substrate (Murray, 1994).
Several SAC proteins are known to associate and these complexes seem to be important for function. Bub3 binds to Bubl through the cell cycle, and this interaction is though to recruit Bubl to kinetochores (Roberts et al., 1994). The association of M adl with Mad2 is necessary for checkpoint function and M adl phosphorylation (Chen et at., 1999). Recent data indicate that following checkpoint activation, Bubl and Bub3 bind to M adl and that formation of this complex is crucial for checkpoint function (Brady and Hardwick, 2000). However, the roles of these complexes remain to be elucidated.
Like the DNA damage checkpoint, the spindle assembly checkpoint arrests cells in metaphase by preventing the function of APC/C^^^^°. However, the SAC arrest is thought to be due to sequestration of Cdc20 (Figure 1.2). In human cells, Mad2 binds to APC/C^'^‘'^° and directly inhibits its activity (Fang et al., 1998), whilst in budding yeast, Cdc20 and Mad2 associate, and cells over-expressing Cdc20 fail to establish a SAC arrest (Hwang et al., 1998). Thus the DNA damage and spindle assembly checkpoints both regulate the metaphase/anaphase transition, by subtly different methods.