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

MSSP（myc single strand binding proteins，myc单链结合蛋白）是一种复制/转录因子，可与C-MYC、cdc激酶等多种蛋白质形成特异性复合物。此外，MSSP直接与DNA复制的主要酶DNA聚合酶α结合，并在体外刺激其活性。在C-MYC、cdc激酶和DNA聚合酶α之间观察到与MSSP的竞争性结合。因此，MSSP在细胞周期从G1期到S期的“许可”过程中发挥了重要作用。对MSSP基因组的分析表明，MSSP基因的表达受启动子上游序列的调控，与C-MYC基因一样，在G1早期被诱导表达，在G1晚期至S早期达到高峰。由于MSSP基因的导入不仅激活了细胞的复制，而且激活了各种启动子的转录，MSSP可能是复制和转录的调控因子。然而，我们所获得的结果表明，MSSP直接参与复制起始的调节，并使细胞与C-MYC一起进入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以及MSSP特异性识别覆盖myc盒的N-末端区域。AMY-1具有反式激活活性，在细胞质侧定位于核膜上并沿核膜沿着，但在C-MYC表达变高的G1晚期和S早期易位入核。因此，这些结果强烈表明，复制因子MSSP和转录因子AMY-1之间的竞争性结合C-MYC的功能作为一个开关的细胞'许可'的S期由C-MYC。

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.