Two birds with one stone

Eukaryotic cells employ a series of elegant regulation mechanisms to replicate the entire genome only once-per-cell cycle to maintain genetic stability. According to the currently accepted model, the replication activation process begins by recruiting an origin recognition complex (Orc1–6) to potential initiation sites during late M-early G1 period. The origin-ORC complex then recruits Cdc6 and Cdt1 to the site, which in turn facilitates the loading of the MCM licensing complex onto an origin to form a pre-replication complex. As a cell progresses further toward S phase many more replication proteins are recruited before the initiation of DNA replication at each origin starts, perhaps by asymmetric bidirectional replication.^1,2 DNA replication is then terminated by the ubiquitination-mediated disassembly of the Cdc45-MCM-GINS helicase complex when 2 converging replication forks meet.³

Once MCM proteins are loaded onto an origin, both Cdc6 and Cdt1 must be inactivated to prevent reloading of the licensing factors. Cdt1 regulation is well described and has been found to be subsequently inactivated either by proteasome-mediated degradation or by binding to geminin. However, the regulation of Cdc6 is not well understood as several seemingly contradictory data have been reported. Certain data supports the idea that Cdc6 is displaced from origin and degraded upon the loading of MCM in G1.⁴ Other data is consistent with the notion that Cdc6 is transported to the cytoplasm during S phase, where it is degraded by an APC-dependent manner.⁵ Still, there is evidence that Cdc6 remains chromatin bound during S phase.⁴ The paper by Kalfalah et al. in this issue of Cell Cycle clarifies this controversy and potentially expands the function of Cdc6 to chromosome segregation at mitosis.⁶ In this study, the authors used time-lapse and photobleaching experiments, dynamic protein stability assays, subcellular fractionations, and the localization of recombinant fluorescently tagged Cdc6. As summarized in Figure 1, the authors found that Cdc6 is degraded by proteasome in the beginning of S phase, followed by a new round of Cdc6 expression in mid- to late-S phase. Interestingly, Cdc6 proteins in these cell cycle compartments are localized to the cytoplasm. These data demonstrate that the degradation and cytoplasmic localization of Cdc6 are temporally regulated separated events. Although microscopic image is clearly in agreement with this conclusion, data from their subcellular fractionation is more complicated, since Cdc6 protein was mainly found with chromatin fraction by 4 hours post-double thymidine. The authors argue that this discrepancy may be an experimental artifact that might have occurred during cell lysis; however, this issue should be clarified in future

Figure 1. The regulation of Cdc6 in the context of cell cycle progression.

Intriguingly, Kalfalah et al. also found that Cdc6 is localized to centrosomes of cells at G2-anaphase.⁶ By telophase, Cdc6 is no longer observed with centrosomes, but localized to the cleavage furrow of the 2 daughter cells. In addition, the authors found that Cdc6 is associated with (condensed) chromatin immediately following the breakage of the nuclear membrane, which lasted until G1 phase of the new cell cycle. This set of data suggests that Cdc6 may have an important role in the segregation of chromosomes, in addition to its essential function in the regulation of loading MCM licensing factors. This is consistent with recent observations that increasing numbers of replication proteins are also involved in mitotic regulation.⁷

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.