Joint study on activation of eukaryotic replication origins

cdc7相关激酶从酵母到人类都是保守的。我们还表明，这些催化亚基的激酶活性受到调控亚基的调节，正如在S.cerevisae中所报道的那样。我们已经通过抗体显微注射实验证明，在哺乳动物细胞中，结合并激活huCdc7激酶的H37的功能是G1向S转化所必需的。H37的表达受生长周期和细胞周期调控，在静止期和G1期低表达，在G1后期升高，在S期保持高表达。MCM2和MCM3是huCdc7/H37激酶复合物的底物。muCdc7 （Cdc7的小鼠同源物）基因的两个等位基因的破坏导致早期胚胎致死，这表明Cdc7功能对哺乳动物的生长和/或早期发育是必需的。对裂变酵母cdc7相关激酶复合物（Hsk1/Him1）的遗传和生化分析揭示了cdc7相关激酶的两个新功能，即对减数分裂前DNA复制的需求，以及对胡诱导的复制叉阻滞和DNA损伤的反应。调控亚基的结构比较揭示了两个保守基序的存在。基序- c对于有丝分裂的功能至关重要，而基序- n对于有丝分裂的正常生长是必不可少的，对于复制分叉阻断或DNA损伤后的恢复可能是必不可少的。在HU阻滞S期后，Him1蛋白发生过磷酸化。我们已经确定了在Him1蛋白的基序n中保守的三个丝氨酸/苏氨酸残基，它们似乎对细胞对复制叉阻断和DNA损伤的反应至关重要。与这些结果一致，him1与先前分离的rad35^+相同。

Cdc7-related kinases are conserved from yeasts to human. We have also shown that kinase activity of these catalytic subunits are regulated by regulatory subunits as has been reported in S.cerevisae. We have shown that the function of H37, which binds and activates huCdc7 kinase, is required for G1 to S transition in mammalian cells by antibody microinjection experiments. Expression of H37 is both growth- and cell cycle-regulated, being low in quiescent as well as in G1 phase, increasing at late G1 and being kept high during S phase. MCM2 and MCM3 are among substrates of huCdc7/H37 kinase complex. Disruption of both alleles of the muCdc7 (mouse homologue of Cdc7) gene resulted in early embryonic lethality, indicating the requirement of Cdc7 function for growth and/or early development of mammals.Genetic and biochemical analyses of fission yeast Cdc7-related kinase complex (Hsk1/Him1) revealed two novel functions of Cdc7-related kinases, namely requirement for premeiotic DNA replication and for response to HU-induced replication fork blocks and DNA damages. Comparison of the structures of the regulatory subunits revealed the presence of two conserved motifs. Motif-C is essential for mitotic functions, while motif-N, dispensable for normal mitotic growth, may be essential for survival after replication fork blocks or recovery from DNA damages. Him1 protein undergoes hyperphosphorylation upon S phase arrest by HU.We have ldentified three serine/threonine residues conserved in the motif-N of Him1 protein that appear to be critical for cellular responses to replication fork blocks and to DNA damages. In consistent with these results, him1 is identical to previously isolated rad35^+.