In this article we have extended the use of fluorescently tagged replication proteins to study the function of replication factors in fission yeast. Cells containing tagged Mcm2–7 and Cdc45 allow two steps leading to S phase to be monitored in single cells, one corresponding to pre-RC formation and the other occurring around DNA replication onset. Cdc45 chromatin association should provide a useful cytological method to distinguish cells in late mitosis/G1 from those in S phase and offers an alternative to methods that cannot be applied to single cells and require synchronization of cell populations.
Using this approach, we have shown that Cdc23 functions after Mcm2 chromatin binding, implying that it is not needed for pre-RC formation, but is necessary for the chromatin association of Cdc45 during replication initiation. This function is conserved between vertebrates and fission yeast, because comparable findings for Mcm10 function have been reported using a soluble in vitro system for DNA replication derived from Xenopus
eggs (Wohlschlegel et al., 2002
). However, in S. cerevisiae
, inactivation of Mcm10 leads to loss of chromatin-associated Mcm2 in G1-arrested cells, leading to the conclusion that Mcm10 is necessary for the earlier step of pre-RC formation (Homesley et al., 2000
). Further work will be required to determine whether this represents an Mcm10 function not conserved in fission yeast or Xenopus
or is a result of differences in experimental design.
What precisely is the biochemical role of Cdc23/Mcm10 in stimulating Cdc45 chromatin binding? One possibility is that it acts as a molecular tether between Cdc45 and other components of the pre-RC, to allow Cdc45 to associate with chromatin. Once loaded, Cdc45 could carry out origin unwinding (Walter and Newport, 2000
) and subsequent assembly of RPA, polymerase α, and polymerase ε during initiation (Mimura and Takisawa, 1998
; Mimura et al., 2000
; Uchiyama et al., 2001
). Cdc45 is required for elongation of replication forks (Tercero et al., 2000
), and Cdc23/Mcm10 could be essential for elongation by maintaining Cdc45 chromatin association during DNA synthesis. However, we find that if cells are arrested in S phase with HU after the Cdc45 chromatin-binding step, and then Cdc23 is inactivated, most cells retain Cdc45 chromatin association. The possibility that incomplete inactivation of Cdc23 is the explanation for a modest reduction in chromatin bound Cdc45 is not supported by the observation that when cells are released from the HU block in this experiment, they fail to complete S phase. This implies that the cells that retain chromatin associated Cdc45 are incapable of completing DNA replication in the absence of Cdc23 function. One interpretation of these results is that Cdc23/Mcm10 is not simply a tether for Cdc45, but affects Cdc45 chromatin association indirectly. For instance, Cdc23/Mcm10 could catalyze a step after pre-RC formation that is needed for both initiation and elongation. Cdc45 could bind as a consequence of this function at initiation, but, once bound, maintenance of its chromatin association would not be so dependent on Cdc23/Mcm10's function during elongation. While this article was under review, Lee et al.
) reported that the in vitro phosphorylation of Mcm2 and Mcm4 by Hsk1 is stimulated by Cdc23. If the critical event for Cdc45 chromatin binding is this Mcm2,4 phosphorylation event, this would explain the dependence of Cdc45 chromatin binding on Mcm4, Hsk1, and Cdc23 reported here.
In spite of the common Cdc23/Mcm10 function between fission yeast and vertebrates, the periodicity of Mcm10 chromatin association during the vertebrate cell cycle contrasts with the constitutive binding of Cdc23 in fission yeast cells. The possible protein interactions that are important for Cdc23/Mcm10 chromatin association are shown in the model in . Chromatin association of fission yeast Cdc23 is shown to occur via ORC and, although direct evidence is lacking, this interaction is plausible based on interactions with ORC subunits in fission yeast (Hart et al., 2002
) and humans (Izumi et al., 2000
) as well as enrichment at origin sequences in S. cerevisiae
(Homesley et al., 2000
). To explain the elongation requirement for Cdc23, Cdc23 is shown departing with the replication forks in association with the putative Mcm2–7 helicase, allowing ORC to bind free Cdc23. Interaction between Cdc23 and Mcm2–7 proteins is suggested by a number of studies (Merchant et al., 1997
; Homesley et al., 2000
; Izumi et al., 2000
; Liang and Forsburg, 2001
; Hart et al., 2002
), although from a consideration of the relative levels of these proteins in fission yeast only a small proportion of total Mcm2–7 can be associated in a complex with Cdc23. An alternative explanation for the function of Cdc23 during elongation that does not require its participation at the replication fork is that its presence at ORC could facilitate the passive replication of unfired origins, as suggested by pausing of forks at origins in a budding yeast mcm10
mutant (Homesley et al., 2000
Figure 9. Model showing possible basis of Cdc23/Mcm10 chromatin association in fission yeast and vertebrate cell cycles. Arrows indicate hypothetical protein-protein interactions that may be important for establishing chromatin association of Cdc23/Mcm10. For details (more ...)
In vertebrates, the main difference compared with fission yeast is that Mcm10 only binds after Mcm2–7 chromatin association, perhaps as interaction with these proteins rather than ORC is important for Mcm10 chromatin binding (). This is consistent with the observation that during S phase, Mcm10 disassociates along with Mcm2–7 proteins from chromatin (Wohlschlegel et al., 2002
). Comparison of Mcm10 sequences reveals that metazoan proteins have a C-terminal extension not found in yeasts (Izumi et al., 2000
), which contains a conserved domain, and it will be of interest to determine whether this is relevant to the distinct chromatin binding properties of vertebrate Mcm10.
These nonconserved patterns of Cdc23/Mcm10 chromatin association during the cell cycle comparing yeasts and vertebrates are intriguingly similar to those seen with Cdc7. S. cerevisiae
Cdc7 is also bound to chromatin throughout the cell cycle (Weinreich and Stillman, 1999
), and the chromatin interaction of the Dbf4 regulatory subunit of Cdc7 is dependent on ORC (Pasero et al., 1999
; Duncker et al., 2002
). In contrast, Xenopus
Cdc7 requires prior binding of Mcm2–7 proteins for its chromatin interaction (Jares and Blow, 2000
). If chromatin associations of Cdc7 and Mcm10, which both function around initiation, are solely dependent on Mcm2–7 proteins in vertebrates, but dependent on ORC in yeasts, this could reflect a dispensability of ORC for initiation in vertebrates once Mcm2–7 chromatin binding has occurred. This is relevant to the consideration of models suggesting that Mcm2–7 complexes in vertebrates may become distributed over a large region of DNA after loading at ORC, before initiation, (Ritzi et al., 1998
; Edwards et al., 2002
), thus potentially allowing initiation away from ORC.
In this work we have established that quiescent fission yeast cells must reestablish Cdc23 chromatin binding as a requirement for DNA replication, and it will be of interest to establish whether this possible coupling between growth and DNA replication has any regulatory significance. We have shown that this event is independent of Mcm2–7 chromatin association and thus does not seem to be related to the discrete binding of Mcm10 that occurs in vertebrate cell cycles after pre-RC formation. Growing fission yeast cells differ from vertebrate cells in that Mcm10 chromatin binding does not have to be re-established after mitosis, and if this step is rate limiting, it is possible that vertebrates thus have an additional regulatory step in G1 to control DNA replication that is not present in yeast. There are precedents for differences in replication control comparing eukaryotes. Yeasts lack vertebrate replication controls involving geminin (Wohlschlegel et al., 2000
; Tada et al., 2001
) and destabilization of ORC1 chromatin association after replication initiation (Mendez et al., 2002
; Sun et al., 2002
), perhaps because unicellular organisms with small genomes can tolerate a lower fidelity of replication control.