times during S phase (Friedman et a l , 1995; Huberman and Riggs, 1968). As it is
essential to ensure that all DNA sequences are replicated precisely once in each cell cycle, none of the large number of the replication origins must fire more than once per cell cycle.
The mechanism ensuring that origins fire no more than once in a cell cycle was extensively studied in a number of species. In the classic set of experiments HeLa cells in different stages of the cell cycle were fused. Fusion of cells in G l with cells in S phase induced DNA replication in the G l nucleus, while fusion of G2 cells with cells in S phase did not induce DNA synthesis in the G2 nucleus (Rao and Johnson, 1970). These experiments indicated that a diffusible S phase activator can only act on G l nuclei which are competent to replicate and not on nuclei in G2.
The recently described pre-replicative complexes assembled on the replication origins in G l in S. cerevisiae can explain the replication potential o f G l, but not G2 nuclei
(Diffley et a l , 1994). Only in G l, pre-replicative complexes are assembled on the
replication origins, and only those origins are competent to fire. Following initiation o f DNA replication from a particular origin, the pre-replicative com plex is disassembled and the origin is incompetent to initiate another round o f DNA replication. New round o f DNA replication can only initiate from this origin following the re-setting of pre-replicative complex.
Attention therefore turned towards understanding the mechanism which allows pre- replicative complexes to assemble on replication origins in G l phase and prevents their re-setting within the same cell cycle. To ensure that DNA replication occurs
precisely once per cell cycle a mechanism must link the process of origin licencing to the control of the cell cycle.
In S. cerevisiae, CDC28 protein kinase is the major regulator of the cell cycle. The
CDC28 protein kinase in association with C lbl-6 B-type cyclins is absolutely required for the initiation of DNA replication as cells in which the Clbs are not
functional fail to enter S phase (Schwob et al., 1994).
Replication origins in cells arrested with nocodazole at the G2/M boundary are normally in a post-replicative state. However, expression of SICl protein, which is a specific inhibitor o f CDC28/Clb kinases caused formation o f pre-replicative complexes in these cells. This suggests that the kinases responsible for origin firing, function to prevent re-setting of the pre-replicative complexes later on in the cell
cycle (Dahmann et al., 1995; Mendenhall, 1993; Schwob et al., 1994). Indeed,
during G 1-phase, when pre-replicative complexes are formed, SICl accumulates and
only disappears shortly before S phase (Schwob et al., 1994). These results provide a
simple model for restricting origin firing to once per cell cycle. The pre-replicative complexes assemble during G l, when CDC28/Clb activity is low. The activation of CDC28/Clb kinase drives cells into S phase, however, the high CDC28/Clb kinase prevents pre-replicative complexes reforming after origin firing. Pre-replicative complexes can only be reset when CDC28/Clb activity drops following the exit from mitosis.
S. cerevisiae CDC6 protein is required for the formation of pre-replicative complexes
and for initiation of DNA replication (Cocker et al., 1996). CDC6 protein can only
promote DNA replication in a restricted window in the cell cycle between the destruction o f Clbs after anaphase and before activation o f Clb5/CDC28 and
C hapter 1
Clb6/CDC28 in late G l (Piatti et a l, 1996). When CDC6 is expressed only later in
the cell cycle, following the activation of the Clb/CDC28 kinases, it is no longer able
to induce pre-replicative complex formation (Piatti et a l , 1996). Conversely, when
Clb2 is expressed in G l, prior CDC6 expression, formation o f pre-replicative complexes is also inhibited (Detweiler and Li, 1998). Presumably, Clb/CDC28 kinases prevent resseting of the replication origins by preventing CDC6 forming pre- replicative complexes.
Experiments in S. pombe provided further evidence that mitotic kinases serve to
prevent re-replication. In S. pombe, CDC2 is the major cell cycle kinase and cdcl3
encodes the single B-type cyclin. Inactivation of CDC2 in cells arrested in G2 and consequent re-activation of the kinase led to re-entry in to S phase rather mitosis
(Broek et a l , 1991). Presumably, pre-replicative complexes re-assembled at the
replication origin during the low kinase activity and subsequent elevation o f the kinase triggered the origin firing. Consistent with this interpretation, deletion of the
CDC13 B-type cyclin also causes re-replication (Hayles et a l , 1994). Over
expression of CDC2/CDC13 protein kinase in cells arrested in G l induced entry into
mitosis, without going through S phase (Hayles et a l , 1994). Finally, overexpression
o f R U M l, a specific inhibitor of CDC2/CDC13 protein kinase, also induced re replication (Correa-Bordes and Nurse, 1995; Moreno and Nurse, 1994).
In Xenopus egg extracts, entry into mitosis is controlled by activation o f CDC2
protein kinase which associates with mitotic cyclins. However, depletion o f CDC2 kinase did not inhibit DNA replication (Fang and Newport, 1991). Instead, a related protein kinase CDK2, which is 66% identical to CDC2 is normally required for the
initiation of S phase. The Xenopus egg extracts from which CDK2 kinase was
replication forks. Re-addition of the CDK2 kinase restored the extracts' ability to initiate DNA replication (Blow and Nurse, 1990; Fang and Newport, 1991).
In Xenopus egg extracts, cyclin E is the major partner of CDK2 (Jackson et a l ,
1995). Immunodepletion of cyclin E from Xenopus egg extract led to the inhibition
o f DNA synthesis and this inhibition was overcome by addition o f cyclin E protein
(Jackson et a l , 1995). Interestingly, addition of cyclin A could also rescue DNA
replication, suggesting that although CDK2/cyclin E is responsible for triggering the initiation of DNA replication, CDK2/cyclin A can substitute. However, addition of
cyclin B was unable to rescue the replication defect (Jackson et a l , 1995).
The CIPl protein is a specific inhibitor of cyclin dependent kinases, with preference
for CDK2/cyclin E and CDK2/cyclin A (Jackson et a l , 1995). CIPl added to
Xenopus egg extract strongly inhibited chromosomal replication, presumably due to
binding and inactivation of CDK2/cyclin E (Chen et a l , 1995; Jackson et a l , 1995;
Strausfeld et a l , 1994). This inhibition could again be relieved by addition of cyclins
A or E, but not cyclin B (Strausfeld et a l , 1994).
The requirement for cyclin dependent kinases for initiation o f DNA replication was
further examined in interphase Xenopus egg extracts from which cdk's and associated
cyclins were depleted. In this assay C DK 2/cyclin E, C D K 2/cyclin A and
CDC2/cyclin A complexes were all able to promote initiation (Strausfeld et a l ,
1996). In a similar set of experiments, both CDC2 and CDK2 were able to rescue
replication (Chevalier et a l , 1995). Experiments in cell free system derived from
HeLa cells confirmed the results obtained using Xenopus egg extracts. G l nuclei in
this system could be induced to enter S phase by CDK2/cyclin E and CDK2/cyclin A
C hapter 1
The emerging view is that CDK2/cyclin E kinase is responsible for initiating DNA replication in higher eukaryotes. Consistent with this idea, cyclin E was found to be
associated with replicating S phase nuclei in the Xenopus system. Under limiting
cyclin E conditions, only the replicating nuclei showed cyclin E staining, suggesting that cyclin E was essential for replication (Chevalier et a l , 1996). While cyclin A
associated kinases can substitute for the CDK2/cyclin E function in promoting S phase, CDK2/cyclin E normally performs this function.
Similarly to the mechanism preventing re-replication in yeast, CDK2/cyclin E and CDK2/cyclin A kinases are implicated in having dual function. In addition to their function in S phase promotion, these kinases prevent replication if they are elevated
s prior proteins essential for DNA replication bind chromatin. In particular, MCM3
protein failed to bind chromatin in the presence of high levels of CDK2/cyclin E (Hua
et a l , 1997). The mechanism which restricts initiation of DNA replication to only
once per cell cycle is therefore likely to be conserved among all eukaryotic systems.