Heichman K A, Roberts J M
Howard Hughes Medical Institute, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Mol Cell. 1998 Feb;1(3):457-63. doi: 10.1016/s1097-2765(00)80046-5.
The Cdc28p cyclin-dependent kinase is thought to both catalyze the onset of DNA replication and prevent rereplication by blocking the reassembly of initiation complexes at replication origins. Budding yeast with mutations in the CDC16 gene represent an exception to this model, because they rereplicate DNA despite being in a G2-like arrest with continually elevated Cdc28p kinase activity. We show, in contradiction to Pichler et al. (1997), that the extra DNA that accumulates in cdc16 mutants is largely chromosomal, as we originally reported. Two-dimensional DNA electrophoresis shows that cdc16 mutants reinitiate DNA synthesis from normal chromosome replication origins, and density transfer experiments show that multiple chromosomal locations are affected. Rereplication from origins requires both Cdc6p and Cdc46/Mcm5p, initiation proteins that had been thought to be inactivated by the Cdc28p kinase. These results establish that CDC16 is required to prevent inappropriate firing of replication origins.
Cdc28p 细胞周期蛋白依赖性激酶被认为既能催化 DNA 复制的起始，又能通过阻止复制起始复合物在复制起点处重新组装来防止再次复制。CDC16 基因发生突变的芽殖酵母是该模型的一个例外，因为尽管它们处于类似 G2 期的停滞状态且 Cdc28p 激酶活性持续升高，但它们仍会再次复制 DNA。与 Pichler 等人（1997 年）的观点相反，我们发现，如我们最初报道的那样，在 cdc16 突变体中积累的额外 DNA 在很大程度上是染色体性质的。二维 DNA 电泳显示，cdc16 突变体从正常染色体复制起点重新启动 DNA 合成，密度转移实验表明多个染色体位置受到影响。从起点再次复制需要 Cdc6p 和 Cdc46/Mcm5p，这两种起始蛋白曾被认为会被 Cdc28p 激酶失活。这些结果表明，CDC16 是防止复制起点不适当启动所必需的。
