Studies on regulation of mitotic DNA replication and meiosis by novel Cdc7-related kinase complexes

Cdc 7激酶及其激活因子Dbf 4蛋白最初在芽殖酵母中发现，在真核生物包括裂殖酵母和人类中广泛保守。Dbf 4相关激活剂（Dfp 1/Him 1和ASK）结合并刺激Cdc 7编码的催化亚基（Hsk 1和huCdc 7）的激酶活性。它的激酶活性是细胞周期调节的，主要通过活化亚基的可用性，其水平在G1/S边界增加，并在整个S期维持在高水平。比较不同真核生物Cdc 7调控亚基的氨基酸序列发现存在三个小的保守氨基酸序列，即Dbf 4-基序-N（BRCT相关）、Dbf 4-基序-M和Dbf 4-基序-C（C2 H2锌指相关）。在体外，含有单独的基序-M或单独的基序-C的小片段与Hskl结合。在体内，一个174个氨基酸的多肽，只含有基序-M（113个氨基酸）和基序-C（61个氨基酸），能够支持himl无效的c 关于我们 细胞以及激酶活化，从而证明Himl与Hskl的二分结合足以用于激酶活化及其体内功能。基序-N，虽然不是有丝分裂功能所必需的，但可能是Himl与染色质相互作用所必需的。在条件性敲除ES细胞中muCdc 7功能的失活导致细胞生长的快速停滞和DNA合成的停止，随后是p53表达的增加和细胞死亡。为了研究小鼠发育中CDK和Cdc 7通路之间的相互作用，我们尝试产生muCdc 7-/-p27-/-双敲除小鼠。在E8.5检测到有活力的胚胎，但此后没有，表明CDK活性的增加可以部分挽救muCdc 7-/-胚胎的早期胚胎生长。MCM 2蛋白是Cdc 7激酶的重要底物之一。MCM 2上的多个残基在体内和体外被Cdc 7磷酸化。我们已经表明，磷酸化的MCM协同行动的Cdks和Cdc 7可能是重要的启动。MCM复合物含有突变体MCM 2缺乏潜在的磷酸化位点的生物化学和遗传特征的哺乳动物和酵母，以澄清Cdc 7介导的起源激活的分子基础。少

Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast are widely conserved in eukaryotes including fission yeast and human. Dbf4-related activators (Dfp1/Him1 and ASK) bind and stimulate kinase activity of Cdc7-encoded catalytic subunits (Hsk1 and huCdc7). Its kinase activity is cell cycle-regulated, mainly through availability of the activation subunit whose level increases at the Gl/S boundary and is maintained at a high level throughout S phase. Comparison of the amino acid sequences of the Cdc7-regulatory subunits from various eukaryotes revealed the presence of three small stretches of conserved amino acid sequences, namely Dbf4-motif-N (BRCT-related), Dbf4-motif-M, and Dbf4-motif-C (C2H2 zinc finger-related). In vitro, a small segment containing motif-M alone or motif-C alone binds to Hskl. In vivo, a 174 amino acid polypeptide containing only motif-M (113 amino acids) and motif-C (61 amino acids) is capable of supporting mitotic growth of himl null c … More ells as well as kinase activation, thus demonstrating that bipartite binding of Himl to Hskl is sufficient for kinase activation and for its functions in vivo. Motif-N, although not essential for mitotic functions, may be required for interaction of Himl with chromatin.Mice lacking muCdc7 genes die between E3.5 and E6.5. Inactivation of muCdc7 functions in conditional knockout ES cells resulted in rapid arrest of cell growth and cessation of DNA synthesis, followed by increase of p53 expression and cell death. In order to examine interactions between CDK and Cdc7 pathways in mouse development, we tried to generate muCdc7-/-p27-/-double knockout mice. Viable embryos were detected at E8.5, but not thereafter, indicating that increase of CDK activity can partially rescue the early embryonic growth of muCdc7-/-embryos. MCM2 protein is among physiologically important substrates of Cdc7 kinase. Multiple residues on MCM2 are phosphorylated by Cdc7 in vivo and in vitro. We have shown that phosphorylation of MCM by concerted actions of Cdks and Cdc7 may be important for initiation. MCM complexes containing mutant MCM2 lacking potential phosphorylation sites are being biochemically and genetically characterized in mammals and yeast in order to clarify molecular basis of Cdc7-mediated origin activation. Less