Initiation of DNA replication needs to be precise to ensure that origins are used only once in each cell cycle. The same is true for the process of replication: the cell needs to ensure that chromatin is replicated only once. As described on the replication page, origins are activated by sequential binding of ORC, Cdc18 (Cdc6) and Cdt1, and MCM proteins. Subsequent binding of Cdc45 activates the origin. But is that all the MCMs do?
The MCMs do much more than assemble the preRC at replication origins. Biochemical data suggest that they convert from an origin assembly factor to a replicative helicase, and they move with the replication fork. Genetic experiments show that they have a function after replication initiation, consistent with a replication helicase. However, cytology indicates that they liberally decorate unreplicated chromatin; perhaps they enable it to be replicated, or mark replication fork termination regions, or protect genome integrity. And they are vastly abundant, exceeding the stoichiometry of origins by 100 fold. In the cartoon below, a diagram is presented of these three models; the origin recognition proteins are shown in green; the replication machinery (polymerases etc) is shown in red, and the MCM proteins are shown in blue. Note that these models are not mutually exclusive (in fact, we know that the first two occur).
MCMs have homology to a wide class of putative DNA-dependent ATPases, and bind together as a hexamer. SV40 T antigen and bacterial DnaB protein are the helicases in their respective systems, and also have hexameric structure. However, in vitro helicase activity is only observed for a complex of Mcm4-6-7. Thus, there are some issues yet to be resolved. (1) All six MCMs are essential in eukaryotes, and evidence suggests that in the normal nucleus, they are present at 1:1:1:1:1:1 stoichiometry. But the in vitro data doesn't account for Mcm2,Mcm3, or Mcm5. (2)MCMs are amazingly abundant, far exceeding the stoichiometry of replication origins. Reducing the dose of MCMs has severe phenotypes, even though the cells can still synthesize DNA, and even though the proteins are still very abundant. (3)Cytological data suggests that MCMs liberally decorate unreplicated chromatin, but do not co-localize with the "replication factories" that contain PCNA and other elongation factors.
So just what are they up to? Recent studies also implicate the MCMs in maintenance of genome integrity and response to damage, and in transcription. These may be multi-purpose factors that have multiple roles beyond their essential function in DNA replication.
Our work has examined the behavior of fission yeast MCMs in vivo, including characterization of the phenotypes associated with temperature sensitive and site-directed mutants, protein associations, and interaction with other factors. Among our other investigations, we have shown that fission yeast MCMs are constitutively located in the nucleus (as it the case for higher eukaryotes as well--only the budding yeast MCMs shuttle in and out of the nucleus during the cell cycle), that intact MCM complexes are required for nuclear localization, ensuring stoichiometry of the complex is maintained in the nucleus, and that high doses of MCM protein are required for genome stabilty. We have also found that the requirement for MCM activity is very different in mitosis and meiosis. Other work in the lab investigates associated factors including the putative MCM kinase Hsk1/CDC7, the chromatin protein Cdc23/MCM10, and the activator Sna41/CDC45.
© S. L. Forsburg .
