Analysis of the cell cycle regulation of chromosome condensation protein complex, condensin.

凝缩蛋白是由SMC异二聚体（SMC2/CAP-E和SMC4/CAP-E）和其他三个非SMC亚基（CAP-D2， -G和-H）组成的保守的五聚体蛋白复合物，在体内有丝分裂染色体凝聚中起重要作用。在体外实验中，凝缩蛋白利用ATP水解的能量在topo I存在的情况下诱导正超旋入DNA。除了有丝分裂功能外，凝缩蛋白还参与间期的染色质调控，如DNA修复、损伤检查点反应和转录调控。我们精确地分析了凝缩蛋白复合物的细胞周期调控。凝缩蛋白亚基的蛋白水平和稳定性在整个细胞周期中几乎是恒定的。凝缩蛋白在有丝分裂期间被Cdc2磷酸化，在间期被CK2磷酸化。与cdc2诱导的凝缩蛋白I磷酸化对超卷曲的刺激作用相反，CK2的磷酸化降低了凝缩蛋白I的超卷曲活性。CK2介导的凝缩蛋白I磷酸化在空间和时间上的调节方式与cdc2介导的磷酸化不同：CK2依赖性磷酸化在间期增加，在有丝分裂期间在染色体上减少。这些发现首次证明了凝缩蛋白I的负调控模式，这一过程可能影响间期和有丝分裂期间的染色质结构。当凝缩蛋白加入体外转录系统后，转录水平降低，抑制作用被ck2介导的磷酸化所抵消。因此，染色质结构可能在间期被凝缩蛋白压紧，ck2介导的磷酸化抑制凝缩蛋白活性导致染色质结构松弛和反活化。

Condensin, a conserved pentameric protein complex composed of SMC heterodimer (SMC2/CAP-E and SMC4/CAP-E) and three other non-SMC subunits (CAP-D2, -G, and -H), plays an essential role in mitotic chromosome condensation in vivo. Condensin induces positive supercoiling into DNA in the presence of topo I using the energy of ATP hydrolysis in vitro. In addition to their mitotic functions, condensins have been implicated in chromatin regulation during interphase, such as DNA repair, damage checkpoint response and transcriptional regulation.We analyzed precisely cell cycle regulation of condensin complex. The protein levels and stabilities of condensin subunits were almost constant throughout the cell cycle. Condensin was phosphorylated by Cdc2 during mitosis, and by CK2 during interphase. In contrast to the stimulatory effect of Cdc2-induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2-mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2-mediated phosphorylation : CK2-dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis.When condensin was added into in vitro transcription system, transcription level was reduced, and the suppression was cancelled by the CK2-mediated phosphorylation. Thus, it is possible chromatin structure was compacted by condensin during interphase, and inhibition of condensin activity by the CK2-mediated phosphorylation leads to relaxation of chromatin structure and transactivation.