Nearly 25 years ago, the restriction point was defined as a period in late G1
subsequent to which a mammalian cell was committed to the replication of its genome (68
). The Rb protein is widely believed to function as a central arbiter of this checkpoint, ensuring the orderly progression of cells into S phase. A large body of evidence indicates that Rb indirectly controls the rate of cell proliferation via its physical or functional interaction with a constellation of transcription factors, including E2F, Elf-1, Sp1/Sp3, myoD, C/EBP, and ATF-2 (for a review, see reference 37
). Such interactions are believed to negatively regulate the transcription of genes required for cell cycle progression and stimulate genes associated with growth inhibition and/or differentiation. Here, we report that Rb can also directly prevent cell cycle progression via its association with MCM7, an essential replication protein and a component of licensing factor.
Suggestions that Rb family members play a role in the negative regulation of DNA replication spring from three previous lines of investigation. First, skeletal muscle cell lines derived from Rb−/−
animals form multinucleated myotubes in vitro and yet are stimulated to synthesize DNA by serum (73
). Consistent with these observations, recent studies with nullizygous animals have shown that (i) nuclei in Rb−/−
myotubes show evidence of endoreduplication and are two to four times larger than those of their Rb+/+
littermates and (ii) Rb−/−
myotubes actively synthesize DNA in vivo (88
). Should the deregulation of DNA replication in Rb−/−
myotubes reflect in part the absence of Rb-MCM7 complexes, one must conclude that Rb-related proteins cannot completely compensate for the loss of this Rb function. In apparent accord with this prediction, inhibition of DNA replication in vitro by GST-p130 is reproducibly less efficient than parallel experiments performed with GST-Rb (Fig. ). It will be of interest to determine if such differences extend to p107 by assessing its relative capacity to inhibit DNA replication. Second, antagonists of Rb family members, such as SV40 large-T antigen, induce unscheduled DNA synthesis and aneuploidy in certain cell types (27
). Whether the binding of viral oncoproteins to Rb-family members results in their dissociation from MCM7 is currently under investigation. Finally, Rb has previously been reported to associate with Purα, a single-stranded DNA-binding protein that binds purine-rich sequences at replication origins (1
). Rb binds to Purα in a phosphorylation-dependent manner via its carboxy-terminal pocket region, and thus it is possible that Rb forms a tripartite complex with MCM7 and Purα at origins of replication. Similarly, additional DNA-binding proteins sequestered within the pocket region of Rb-related proteins could be envisioned to participate in the targeting of MCM complexes to euchromatin.
The genome is replicated only once during each cell cycle in most eukaryotic cells, and many of the regulatory mechanisms governing the initiation of DNA replication have been identified by using cell extracts prepared from Xenopus
). DNA added to such extracts is assembled into functional nuclei that undergo a single round of semiconservative replication. To undergo a subsequent round of replication in vitro, either Xenopus
nuclei have to progress through mitosis or the nuclear membrane must be transiently permeabilized. To account for these results, Blow and Lasky proposed that chromosomes are licensed to undergo DNA replication once during each cell cycle by a regulator termed replication licensing factor (RLF) (3
). Binding of RLF to chromatin was proposed to mark a replication origin for firing, and replication was thought to inactivate RLF, ensuring that a single round of DNA synthesis ensues. Following the dissolution of the nuclear envelope in mitosis, chromosomes were predicted to once again become licensed for replication via the binding of RLF to chromatin. Recently, RLF has been resolved into two components, both of which are required for DNA replication (9
). One component appears to facilitate the association of RLF with chromatin, and the other is a protein complex that contains MCM2 through -7 (47
). MCM proteins were originally identified in Saccharomyces cerevisiae
as functions necessary for the maintenance of minichromosomes carrying certain types of autonomously replicating sequences (58
). Such functions have been well conserved throughout evolution, since MCM genes have been isolated from a wide variety of eukaryotes, including plants, insects, amphibians, and mammals (10
). MCM proteins are tightly associated with chromatin until replication begins, whereupon they are released into the nucleoplasm and rebound to chromatin during mitosis. Indeed, this fluctuation in their subcellular localization provided the first indication that MCM proteins might function as a component of RLF.
Although MCM proteins have been identified as components of RLF, questions regarding the regulation of RLF function remain unresolved. For example, what prevents chromatin-associated MCM complexes from triggering DNA replication at inappropriate times? A possibility suggested by results reported here is that the activity of MCM complexes are regulated via their association with Rb family members. This association with MCM7 is mediated by the amino termini of Rb family members, a relatively unexplored portion of these proteins that has previously been shown to be unnecessary for interactions with transcription factors such as E2F (37
). Interestingly, the association of Rb-related proteins with MCM7 requires a portion of MCM7, the carboxy-terminal 137 amino acids, that is not conserved among MCM family members. Thus, Rb, p107, and p130 appear to have evolved to interact specifically with one component of RLF. Although we have shown that Rb interacts with MCM7 in a yeast two-hybrid screen, in in vitro protein-binding assays, in mammalian cells, and in Xenopus
extracts, it is worth noting that our experiments do not prove that these interactions are direct. It remains formally possible that one or more evolutionarily conserved proteins function as a bridge between Rb family members and MCM7. Additionally, our experiments do not address whether Rb family members prevent MCM7 from joining RLF or whether Rb family members are tethered to RLF via their association with MCM7. Since MCM7 is invariably found associated with other MCM proteins in vivo, the latter possibility may be more likely (28
Inclusion of GST-Rb or p130 fusion proteins in Xenopus
extracts resulted in a marked reduction in DNA replication. Importantly, this negative regulation of in vitro DNA replication appears to be MCM7 dependent, since preincubation of Rb and p130 with the carboxy-terminal 137 amino acids of MCM7 (MBP-MCM7c) restored the replicative capacity of Xenopus
extracts. At least two possible mechanisms may account for the capacity of MCM7c to restore DNA replication. First, MCM7c could supplant the loss of XMCM7 function by directly interacting with RLF. Although this is a formal possibility, it is unlikely, since the amino-terminal 122 amino acids of MCM7 are required for interactions with components of licensing factor (72
). A more likely scenario is that preincubation of Rb and p130 with MCM7c saturated MCM7 binding sites, thereby preventing their interaction with XMCM7. It is worth noting that our results do not enable us to formally rule out the possibility that inclusion of MCM7c prevents the interaction of Rb and p130 with yet another Xenopus
protein required for replication. Regardless of the precise mechanism of rescue, results from in vitro replication assays strongly support the idea that Rb family members can negatively regulate DNA replication. Taken together with in vitro and in vivo evidence of protein complexes between Rb, p107, or p130 and MCM7, we conclude that MCM7, and by extension RLF, is a target of tumor suppressor gene function. Interestingly, the product of yet another tumor suppressor gene, p53, has been reported to negatively regulate the initiation of DNA replication in vitro (13
). Whether MCM7 function is directly or indirectly influenced by p53 has yet to be established.
Given the biochemical and functional results reported here, we hypothesize that the initiation of DNA replication is prevented, at least in part, by the association of Rb family members with MCM7 in G1
. Rb, p107, and p130 are phosphorylated by cyclin-cdk kinases as cells approach the G1
/S transition, and we speculate that phosphorylation may trigger the release of MCM7 (83
). Since Xenopus
extracts carry active cyclin-cdk kinases, why do Rb and p130 proteins block in vitro DNA replication? As we have previously reported, the Rb amino terminus is not bound by cyclin-cdk kinases and is only marginally phosphorylated by such complexes in vitro (78
). Indeed, interactions of Rb family members with cyclin-cdk kinases requires their respective pocket regions, a domain not included in the fusion proteins that we have examined (17
). As the cell cycle progresses, dephosphorylation of Rb family members in anaphase may facilitate their reassociation with MCM7 and inhibition of DNA replication until passage through the restriction point during the subsequent cell cycle. Experiments to determine the abundance of complexes between Rb family members and MCM7 as a function of cell cycle progression are currently under way.