6 F.-Y. Bouget et al.
3
Evidence for Different Types of Circadian Checkpoints
The CDC core cell cycle machinery is well conserved in eukaryotes, relying
mainly on Cyclin-dependent-kinases (CDKs) which are required for the main
stages of CDC progression including DNA replication during the S phase and
segregation of chromosomes at mitosis (Inze 2005). CDKs are positively reg-
ulated by association to cyclins and negatively regulated by small inhibitors
(CKI). CDKs are also strongly regulated by phosphorylation and dephos-
phorylation. For example, in G2 the WEE1 kinase down-regulates mitotic
CDKs by phosphorylation whereas the antagonistic CDC25 phosphatase ac-
tivates CDK at the G2/M transition. CDKs are the main targets of the various
checkpoints, which ensure that CDC progression arrests if something wrong
happens such as incomplete DNA replication, DNA damage or spindle de-
fect (Millband et al. 2002; Weinert 1998). Another critical checkpoint is the
cell-size checkpoint also called “sizer” (Kellogg 2003). During development,
cell growth and division must be tightly coordinated to maintain a specific
size. This size control is well known in yeasts. In budding yeast, a critical
restriction point START or (R) has been defined in G1, when cells become
irreversibly engaged in cell division when they reach a critical size. In con-
trast fission yeast grow mainly in G2 and the control of CDC progression
is exerted at the G2/M transition (Kellogg 2003). Photosynthetic organisms
rely on light as the source of energy, to reach a critical size for cell divi-
sion. The regulation of cell division by light was dissected inChlamydomonas.
Two points were defined: in early G1, a restriction point called primary arrest
(A) when cells become light-dependent; in late G1, a “transition point” (T)
when cell division becomes independent of light (Spudich and Sager 1980).
Though it remains to be demonstrated clearly that gating of cell division oc-
curs before entry into the S phase inChlamydomonas,itislikelytobeso,
since arrests outside of the G1 phase are never observed when cells are moved
from light to darkness (Fig. 1). In contrast, light-dependent restriction mech-
anisms were shown to exist both in G1, S and G2 phases of the cell cycle in
Euglenacells transferred from light to darkness (Hagiwara et al. 2002) and
a circadian gating of CDC progression has been observed from G2 to mito-
sis but also at the S/G2 and G1/S transitions in this organism (Bolige et al.
2005). The targets of the circadian checkpoints remain elusive inEuglenaor
Chlamydomonas. The only demonstration of a direct regulation of cell divi-
sion by the circadian clock come from animals. In hepatocytes re-entering
cell cycle upon hepatectomy, the circadian clock gates cell cycle progression at
the G2/M transition, through a direct transcriptional regulation of the WEE
kinase (Matsuo 2003). A negative regulation of cell cycle by the circadian
clock at the G1/S transition was demonstrated in osteoblasts where the clock
inhibits the G1 cyclin D1 (Fu et al. 2002). In summary, the circadian clock ap-