Joint study on DNA replication and checkpoint control

我们一直在广泛地研究G1/S转换的分子机制及其调控。这些研究的结果概括如下：1)CDC7蛋白及其激活物Dbf4蛋白最初在芽殖酵母中发现，在真核生物中广泛保守，包括分裂酵母和人类。我们已经通过产生条件敲除ES细胞证明了CDC7功能对于哺乳动物细胞的DNA复制和增殖活动是必不可少的。2)比较不同真核生物的CDC7调节亚基的氨基酸序列发现存在三个小范围的保守氨基酸序列，即Dbf4-Motif-N(BRCT相关)，Dbf4-Motif-M和Dbf4-Motif-C(C2H2锌指相关)。在体外，一小段单独含有Motif-M或Motif-C的片段与Hsk1结合。在体内，只含有Motif-M(113个氨基酸)和Motif-C(61个氨基酸)的174个氨基酸的多肽能够支持h…的有丝分裂生长更多的IM1缺失细胞以及蛋白激酶的激活，从而证明Him1与Hsk1的两段结合足以激活蛋白激酶及其在体内的功能。Motif-N虽然对有丝分裂功能不是必需的，但可能是Him1与染色质相互作用所必需的。3)缺乏mucc7基因的小鼠在E3.5和E6.5之间死亡。为了研究CDK和CDC7通路在小鼠发育过程中的相互作用，我们尝试产生了muCDC7-/-p27-/-双基因敲除小鼠。在E8.5时检测到有活力的胚胎，但之后没有检测到，这表明CDK活性的增加可以部分挽救muCDC7-/-胚胎的早期胚胎生长。4)我们在人类染色体5q上GM-CSF基因下游的IL-3/GM-CSF细胞因子簇区定位了一个复制起点。此外，我们还发现ORC、CDC6和MCM蛋白与该区域有特异性结合。通过与其他已知的后生动物复制起始序列的比较，我们确定了一个可能的一致序列。较少

We have been extensively studying the molecular mechanisms of G1/S transition and its regulation. The results obtained from these studies can be summarized as follows1) Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast are widely conserved in eukaryotes including fission yeast and human. We have demonstrated that Cdc7 functions are essential for DNA replication and proliferation activities of mammalian cells by generating conditional knockout ES cells.2) 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 Hsk1. 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 h … More im1 null cells as well as kinase activation, thus demonstrating that bipartite binding of Him1 to Hsk1 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 Him1 with chromatin.3) Mice lacking muCdc7 genes die between E3.5 and E6.5. 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.4) We have located a replication origin in the IL-3/GM-CSF cytokine cluster region on the human chromosome 5q at the region immediately downstream of GM-CSF gene. Furthermore, we showed that ORC, Cdc6 and MCM proteins are specifically bound to this region. Through comparison with other known metazoan replication origin sequences, we have identified a possible consensus sequence. Less