Joint study on activation of eukaryotic replication origins

cdc 7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调节调节亚基已在酿酒酵母中报道。我们已经表明，H37的功能，结合和激活huCdc 7激酶，需要在哺乳动物细胞中的G1到S的过渡，通过抗体显微注射实验。H37的表达受生长和细胞周期调节，在静止期和G1期低，在G1晚期增加，在S期保持高水平。MCM 2和MCM 3是huCdc 7/H37激酶复合物的底物。muCdc 7的两个等位基因的破坏Cdc 7（小鼠Cdc 7同源基因）基因的克隆导致了早期胚胎死亡，表明Cdc 7功能对哺乳动物的生长和/或早期发育是必需的。裂殖酵母Cdc 7相关激酶复合物的遗传和生化分析（Hsk 1/Him 1）揭示了Cdc 7相关激酶的两个新功能，即需要减数分裂前DNA复制和响应HU诱导的复制叉阻断和DNA损伤。调节亚基的结构的比较揭示了两个保守的基序的存在。基序-C对有丝分裂功能是必不可少的，而基序-N，对正常的有丝分裂生长是必需的，可能是复制叉阻断后的生存或从DNA损伤中恢复所必需的。Him 1蛋白在被HU阻滞于S期后发生高度磷酸化，我们在Him 1蛋白的基序-N中发现了三个保守的丝氨酸/苏氨酸残基，这三个残基对于细胞对复制叉阻断和DNA损伤的反应至关重要。与这些结果相一致的是，him 1与先前分离的rad 35 ^+完全相同。

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^+.