MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, Sussex BN1 9RR, UK
* Author for correspondence (e-mail: a.m.carr{at}sussex.ac.uk)
The DNA integrity checkpoints ensure completion of DNA replication and DNA
repair before entry into mitosis (O'Connell
et al., 2000). Unreplicated DNA or unrepaired DNA will cause
catastrophic chromosome segregation. The accompanying schematic shows the
checkpoint proteins that monitor the state of DNA replication and DNA
repair.
|
Checkpoint proteins most probably interact with the replication complex
(RC) during S phase (blue wedge) or with DNA strand breaks if they are induced
during G2 phase (yellow wedge). These checkpoint proteins ultimately
inactivate the Cdc2-cyclin-B kinase complex by inhibitory Tyr15
phosphorylation (O'Connell et al.,
2000). High Cdc2-cyclin-B kinase activity allows the cell to enter
mitosis (pink wedge), where chromosome separation and segregation occur (see
bottom left, upper panel). Checkpoint activation thus causes cell cycle arrest
in G2 phase (see bottom left, lower panel). Evolutionary conserved checkpoint
proteins (see table) have been classified into several categories, including
the ATR-ATRIP kinase complex (Rad3-Rad26), the RFC- and PCNA-like complexes
(Rad1, Rad9, Hus1 and Rad17), the mediator proteins (Crb2 and Mrc1) and the
effector kinases (Chk1 and Cds1).
In fission yeast, the ATR-ATRIP complex (Rad3-Rad26) phosphorylates many of
the other checkpoint proteins, and these events are central to the checkpoint
response. Rad3-dependent Chk1 and Cds1 phosphorylation is essential for
checkpoint activation (Rhind and Russell,
2000). Rad17, together with RFC2-RFC5, forms a
replication-factor-C-like complex and is thought to load a PCNA-like
Rad1-Hus1-Rad9 complex onto damaged DNA
(Caspari and Carr, 2002
). This
complex may link Rad3-Rad26 to the mediator complexes, which in turn bring
Chk1 and Cds1 into proximity. Different mediator complexes function in S phase
and G2 phase. In S phase, Mrc1 mediates Cds1-dependent checkpoint signaling.
Activation of Cds1 kinase results in Mik1 accumulation. Mik1 phosphorylates
Tyr15 of Cdc2, thus preventing mitosis. Importantly, the Mrc1/Cds1 module has
additional roles in the S phase response, such as stabilizing stalled
replication forks and inhibiting late-firing replication origins. It also
regulates the Mus81-Eme1 endonuclease complex, which was identified through
its interaction with the Cds1 fork-head-associated (FHA) domain
(Boddy et al., 2000
). In G2
phase, Crb2 mediates Chk1-dependent checkpoint signaling. Activated Chk1
results in activation of Mik1 and Wee1 Cdc2 Tyr15 kinases and may inactivate
Cdc25 tyrosine phosphatase (O'Connell et
al., 2000
).
Chk1 is also involved in regulating dNTP synthesis
(Liu et al., 2003). Chk1
activation in G2 induces degradation of the Spd1 protein. Spd1 behaves as an
inhibitor of ribonucleotide reductase (RNR). Its ubiquitin-dependent
degradation is necessary for nuclear export of Suc22 (the small subunit of
RNR) during DNA replication in S phase (upper right) and DNA repair in G2
phase. Suc22, which normally localizes to the nucleus in G2 phase cells
(bottom right) and the whole cell during normal S phase, presumably forms an
active complex with cytoplasmic Cdc22 during DNA replication and in response
to checkpoint activation to provide nucleotides for DNA repair. The
signalosome complex promotes ubiquitin-dependent degradation of Spd1. The
signalosome forms a complex together with the Pcu4 (cullin 4) E3 ubiquitin
ligase.
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References |
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Boddy, M. N., Lopez-Girona, A., Shanahan, P., Interthal, H.,
Heyer, W. D. and Russell, P. (2000). Damage tolerance protein
Mus81 associates with the FHA1 domain of checkpoint kinase Cds1.
Mol. Cell Biol. 20,
8758-8766.
Caspari, T. and Carr, A. M. (2002). Checkpoints: how to flag up double-strand breaks. Curr. Biol. 12, R105-R107.[CrossRef][Medline]
Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M. and
Caspari, T. (2003). Cop9/signalosome subunits and Pcu4
regulate ribonucleotide reductase by both checkpoint-dependent and -
independent mechanisms. Genes Dev.
17,
1130-1140.
O'Connell, M. J., Walworth, N. C. and Carr, A. M. (2000). The G2-phase DNA-damage checkpoint. Trends Cell Biol. 10, 296-303.[CrossRef][Medline]
Rhind, N. and Russell, P. (2000). Checkpoints: it takes more than time to heal some wounds. Curr. Biol. 10, R908-R911.[CrossRef][Medline]
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