Characterization of Cdc7 functional domains

细胞分裂过程受到严格控制，并与其遗传物质(DNA)的复制密切协调。这一过程的错乱可能是致命的或导致遗传不稳定。被称为CDC7的蛋白激酶通过连接细胞周期引擎(即细胞周期蛋白依赖的激酶)和复制机制(即复制DNA的酶)在这一调节中发挥关键作用。为了执行其功能，CDC7必须进入细胞核，然后与DNA复制开始的染色质(即DNA)结合。通过突变分析，我们先前发现203-370氨基酸片段包含了CDC7核输入的信号。我们还发现，306-326片段包含了CDC7核保留的信号，可能是通过与新DNA合成开始的染色质结合来实现的。这种核滞留信号显然被CDC7蛋白羧基末端的两个小片段所拮抗，我们统称这两个片段为核输出序列。目前，我们还不知道：(A)核输入和保留的信号是一个整合的还是两个独立的元件；(B)核保留序列的21个氨基酸片段(306-326)如何调节CDC7与DNA的相互作用(DNA合成可能被激活或失活)；以及(C)CDC7的氨基酸修饰如何参与调控CDC7与染色质的相互作用。我们在这里建议对CDC7蛋白结构域和每个结构域中的关键氨基酸残基进行深入研究。这项工作的数据将为DNA复制的调控提供重要的新线索，并将有助于解开细胞如何维持遗传稳定性的调控机制。

The cell division process is tightly regulated and closely coordinated with copying of its genetic material (DNA). Derangement of this process can be fatal or result in genetic instability. Protein kinase known as Cdc7 plays a critical role in this regulation by connecting the cell cycle engine (i.e., cyclin-dependent kinase) and the replication machinery (i.e., the enzymes copying DNA). To perform its function, Cdc7 must enter the nucleus and then bind to chromatin (i.e., DNA) where DNA replication starts. By mutation analysis, we previously found that the amino acid segment 203-370 contains the signal for the Cdc7 nuclear import. We also found that the segment 306-326 contained a signal for the Cdc7 nuclear retention, probably by binding to chromatin where new DNA synthesis begins. This nuclear retention signal is apparently antagonized by two small segments at the carboxyl terminus of Cdc7 protein, which we collectively term a nuclear export sequence. At present, we do not know: (a) if the signals for the nuclear import and retention are one integrated or two separate elements; (b) how the 21-amino acid segment (306-326) of the nuclear retention sequence regulates the interaction of Cdc7 with DNA (by which DNA synthesis may be activated or inactivated); and (c) how the amino acid modifications of Cdc7 is involved in the regulation of Cdc7 interactions with chromatin. We here propose to carry out an in-depth study of Cdc7 protein domains and critical amino acid residues within each domain. Data from this work will shed important new light on the regulation of DNA replication, and will help to unravelling the regulation mechanism as to how a cell maintains genetic stability.