The pre-RC is assembled over and around the ORC-binding site at origins of DNA replication in budding yeast. PreRC formation and maintenance require both Cdc6 (Cocker et al., 1996
; Santocanale and Diffley, 1996
) and the MCM2–7 complex (this study). As Cdc6 is required for the association of MCM2–7 proteins with chromatin, and as pre-RC formation cannot occur in cells containing Cdc6 but not MCM proteins, our data suggest that the prereplicative footprint represents the binding site of MCM2–7 proteins alongside ORC.
MCM2–7 proteins have been estimated to be between 20 and 100 times more abundant than ORC, Cdc6, or the number of origins of DNA replication (Lei et al., 1996
; Donovan et al., 1997
), and a significant proportion is associated with chromatin during G1-phase. The reason for the high relative-abundance of MCM proteins remains unclear. It is interesting to note that a mutant allele of CDC6
, supports the formation of a partial prereplicative footprint at ARS305 but does not support the loading of wild-type levels of MCM2–7 proteins onto chromatin (Perkins and Diffley, 1998
). The partial pre-RC induced by Cdc6-d1 protein produces suppression of ORC-induced hypersensitive sites 1 and 2 (see Figures and ) but does not cause suppression of the third ORC-induced hypersensitive site or protection of the adjacent region from DNase I digestion (Perkins and Diffley, 1998
). Because all aspects of the pre-RC footprint at ARS305 are MCM-dependent, it is possible that the partial pre-RC and the full pre-RC differ quantitatively in the number of MCM2–7 complexes bound to the origin. For example, the partial pre-RC may contain a single MCM2–7 complex, and generation of the full pre-RC at ARS305 may require the binding of multiple MCM2–7 complexes. It is worth noting that, at a very late and normally silent origin such as ARS301, the pre-RC footprint involves suppression of two ORC-induced hypersensitive sites without significant protection of adjacent regions (Santocanale and Diffley, 1996
). Perhaps one important difference between early origins, such as ARS305 and very late origins, such as ARS301, is that the former are bound by more MCM2–7 complexes than the latter. It is likely, however, that other factors also contribute to the determination of origin-timing, as proximity to a telomere delays activation of a normally-early origin without changing the prereplicative footprint (Santocanale and Diffley, 1998
Our data, together with those of Nguyen et al.
(Nguyen et al., 2000
), indicate that budding yeast MCM2–7 proteins interact with each other in vivo, during late mitosis, G1-phase, and after S-phase. Furthermore, we show that this interaction is essential for nuclear accumulation of MCM proteins during the period of the cell cycle when they are assembled into pre-RCs, as previously reported for fission yeast (Pasion and Forsburg, 1999
). We have also shown previously that Mcm2, 3, 4, 6, and 7 proteins are required during S-phase for the elongation phase of chromosome replication (Labib et al., 2000
). Taken together, these experiments reinforce the notion that the active form of MCM2–7 proteins, throughout the cell cycle, is likely to be a heterohexamer.
Our experiments show that, once replication forks have been established from early origins, MCM2–7 proteins, and therefore pre-RCs, are not required to inhibit anaphase in response to incomplete chromosome replication. Inhibition of the progression of DNA replication forks, either by HU treatment or by MCM-depletion after initiation, blocks entry into anaphase. In both cases, hyperphosphorylation of Rad53 is maintained, and anaphase is inhibited in a Rad9-independent manner. It appears that stalling of replication forks, rather than presence of MCM2–7 proteins, or pre-RCs, is important for the checkpoint inhibiting mitosis in response to incomplete replication.
Several studies, however, have reported that other replication proteins, such as RF-C (Sugimoto et al., 1997
; Noskov et al., 1998
; Reynolds et al., 1999
; Shimada et al., 1999
) or the budding yeast Dpb11 protein and its fission yeast homologue Cut5 (Saka and Yanagida, 1993
; Saka et al., 1994
; Araki et al., 1995
; McFarlane et al., 1997
; Wang and Elledge, 1999
) are required to maintain checkpoint inhibition of mitosis in HU-arrested cells, suggesting that these proteins may indeed play a role in checkpoint control. But it remains to be shown that activation of early origins and replication fork establishment have occurred normally in these experiments. Failure to establish replication forks, due to the combination of HU and the defective nature of a particular conditional allele chosen for such an experiment could cause entry into anaphase without the test protein having a direct role in checkpoint control.
It is worth noting that mitosis occurs with very similar timing, both in wild-type cells, and also in cells that segregate their chromosomes in the absence of DNA replication (this study, Piatti et al., 1995
; Tercero et al., 2000
). It is likely, therefore, that the timing of anaphase in budding yeast is determined by a second mechanism, distinct from the checkpoint that blocks mitosis in response to incomplete chromosome replication.
We favor the view that some aspect of the structure of replication forks may be sensed by checkpoint proteins, leading to the generation of the checkpoint signal. It has been argued that this may involve detection of the RNA primer present at the beginning of Okazaki fragments (Michael et al., 2000
), but this view is not consistent with experiments implicating RF-C in checkpoint control, as RF-C acts after primer formation, and it is not clear how mutation of RF-C would affect the presence or absence of RNA primers in Okazaki fragments.
Our experiments suggest one approach to addressing these issues in the future, by making degron mutants of other replication proteins such as primase and RF-C and by comparing the effects of degrading these proteins in HU-arrested cells after confirming that establishment of replication forks from early origins has indeed taken place.