MOLECULAR MECHANISMS OF THE CELL CYCLE REGULATION BY REPLICATION/TRANSCRIPTION FACTORS,MSSP

MSSP，myc单链结合蛋白，已被确定为复制/转录因子，并与包括C-MYC和cdc激酶在内的多种蛋白形成特异性复合物。此外，MSSP直接结合DNA聚合酶α，这是DNA复制的主要酶，并在体外刺激其活性。观察到C-MYC、cdc激酶和DNA聚合酶α与MSSP的竞争性结合。因此，MSSP被认为在细胞周期从G1期到S期的“许可”过程中发挥重要作用。对基因组MSSP基因的分析发现，MSSP的表达受启动子上游序列的调控，其表达在G1期早期被诱导，并在G1晚期至S期早期达到与C-MYC相同的峰值。由于将MSSP引入细胞，不仅可以激活复制，还可以激活各种启动子的转录，因此MSSP被认为是复制和转录的调节因子。然而，我们获得的结果表明，MSSP直接参与复制起始的调控，并使细胞与C-MYC一起“获准”进入S期，随后转录被激活或抑制。另一个C-MYC结合蛋白AMY-1（与C-MYC相关）已通过酵母双杂交系统克隆。AMY-1和MSSP特异性识别覆盖myc盒子的n端区域。AMY-1具有反激活活性，定位于细胞质侧核膜上及沿核膜方向，但在G1晚期和S早期C-MYC高表达时易位入核。因此，这些结果强烈表明，复制因子MSSP和转录因子AMY-1之间与C-MYC的竞争性结合是C-MYC“许可”细胞进入S期的开关。

MSSP,myc single strand binding proteins, have been identified as replication/transcription factors and shown to form specific complex with various proteins including C-MYC and cdc kinase. In addition, MSSP directly bound to DNA polymerase alpha, which is a major enzyme of DNA replication, and stimulated its activity in vitro. Competitive binding to MSSP was observed among C-MYC,cdc kinase and DNA polymerase alpha. MSSP was thus suggested to play an important role in the 'licencing' process at the progression from the G1 phase to the S phase of the cell cycle. The analyzes of the genomic MSSP gene revealed that the expression of MSSP was regulated according to the cell cycle by the sequences upstreem from the promoter and the expression was induced in the early G1 phase and reached a peak from the late G1 to the early S phase as that of C-MYC.Since the introduction of MSSP to cells resulted in activation not only of replication but also of transcription from various promoters, MSSP was suggested to be regulatory factors for both replication and transcription. The results we have obtained, however, indicate that MSSP is directly involved in the regulation of replication initiation and make cells 'licensed' to enter the S phase together with C-MYC,and that the transcription is subsequently activated or repressed. Another C-MYC binding protein, AMY-1 (associated with C-MYC) , has been cloned by the yeast two-hybrid system. AMY-1, as well as MSSP,specifically recognizes the N-terminal region covering the myc boxes. AMY-1 has a transactivation activity and localizes on and along the nuclear membrane on the cytoplasmic side, but translocates into nucleus in the late G1 and the early S phase when the C-MYC expression becomes high. These results hence strongly suggest that the competitive binding to C-MYC between the replication factor MSSP and the transcription factor AMY-1 functions as a switch of the cell 'licencing' to the S phase by C-MYC.