Kalfalah Faiza M, Berg Elke, Christensen Morten O, Linka René M, Dirks Wilhelm G, Boege Fritz, Mielke Christian
a Institute of Clinical Chemistry and Laboratory Diagnostics; University Düsseldorf; Medical Faculty , Düsseldorf , Germany.
Cell Cycle. 2015;14(11):1704-15. doi: 10.1080/15384101.2014.1000182.
To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.
为维持基因组稳定性，哺乳动物基因组中的数千个复制起点必须在每个细胞周期仅起始一次复制。这通过在S期正在进行的复制与前一个G1期预复制复合物的形成在时间上严格分离来实现，预复制复合物“许可”每个复制起点具备复制能力。然而，由于关于Cdc6的稳定、降解和核输出存在看似矛盾的发现，装载因子Cdc6对许可过程时间安排的贡献仍然难以捉摸。利用在活的循环细胞中表达的荧光标记Cdc6（Cdc6-YFP），我们在此证明Cdc6-YFP在有丝分裂和G1期是稳定的且与染色质相关。它在S期起始时经历快速的蛋白酶体降解，随后在S期和G2期被主动转运到细胞质中。生化分级分离消除了这种核排斥，导致Cdc6-YFP以及可能的内源性Cdc6与染色质异常结合。此外，我们证明Cdc6在G2晚期和有丝分裂期间与中心体相关。这些结果表明多种Cdc6调节机制共存，但以细胞周期特异性方式受到严格控制。
