Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

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，哺乳动物基因组的数千个复制起点必须在每个细胞周期仅启动一次复制。这一过程通过严格的时间分隔实现：S期进行活跃的DNA复制，而在前述G1期形成前复制复合物（pre-replicative complexes），后者可"许可"每个复制起点获得复制能力。然而，由于针对Cdc6（细胞分裂周期蛋白6，Cdc6）的稳定性、降解及核输出的研究结果看似相互矛盾，其作为加载因子对复制许可过程时序的调控贡献始终难以明确。本研究通过在增殖活细胞中表达荧光标记的Cdc6（Cdc6-YFP，YFP即黄色荧光蛋白（yellow fluorescent protein）），证实Cdc6-YFP在有丝分裂期及G1期保持稳定且与染色质结合；其在S期起始阶段会经历快速的蛋白酶体降解，随后在S期及G2期主动输出至细胞质。生化分级分离操作会破坏这种核输出调控机制，导致Cdc6-YFP乃至内源性Cdc6出现异常的染色质结合。此外，本研究证实Cdc6在G2晚期及有丝分裂期与中心体存在结合。上述结果表明，Cdc6存在多种调控机制，但这些机制均以细胞周期特异性的方式受到严格管控。