Studies of molecular mechanism on assembly of mammalian pre-replicative complex and replication apparatus

将起源识别复合体(ORC)装载到染色质上对于组装前复制复合体是必不可少的。为了研究哺乳动物ORC的生理功能，我们从小鼠NIH3T3细胞中克隆了ORC亚单位的cDNA，并发现了ORc1、Orc2和OrC3的新变体，每个变体都是通过选择性RNA剪接产生的。Orc1A和Orc1B的特征使我们能够确定ORC1的结构域，它是核转位、磷酸化和降解所必需的。为了进一步阐明Orc1A磷酸化的作用，我们制备了一种针对含有Ser^&lt；276&gt；的磷酸肽的抗体，Orc1A蛋白在G2/M期主要被磷酸化。此外，在S期，少量磷酸化的Orc1a逐渐增加，而大多数Orc1a保持非磷酸化，并与染色质结合。所有被磷酸化的orc1a都是从含有100mMNaCl染色质未结合部分中提取出来的，瞬时表达的orc1a是部分磷酸化的…当核转位缺陷的ORC1突变体被广泛磷酸化时，我们发现Ser^&lt；276&gt；的磷酸化依赖于保守的周期蛋白对接(Cy)基序，该基序位于氨基末端溴相邻同源(BAH)结构域的旁边。ORC1BAH结构域在酿酒酵母中与Sir1结合，在果蝇和人类中作为HP1结合。综上所述，我们得出结论，小鼠ORC1至少在整个细胞周期中受到Ser^和276&gt；残基磷酸化的调控。在G1期，未磷酸化的Orc1a与染色质结合，主要定位在异染色质上。在S期，Orc1A逐渐从染色质中释放出来，并被CDK/Cyclin A通过Cy基序磷酸化。磷酸化的ORc1在S期降解。相反，在G2/M期，磷酸化的ORC1没有被降解，而是在去磷酸化后与异染色质重新结合。我们目前正在试图了解Cy基序是否通过与HP1或其他染色质因子相互作用而被染色质上的氨基末端BAH结构域所掩盖。较少

Loading of the origin recognition complex (ORC) onto the chromatin is essential for the assembly of the pre-replicative complex. To examine physiological functions of the mammalian ORC, we cloned cDNAs of Orc subunits from mouse NIH3T3 cells and found novel variant forms of Orc1, Orc2, and Orc3, each generated by alternative RNA splicing. Characterization of Orc1A and Orc1B allowed us to identify the domain of Orc1, which is essential for nuclear translocation, phosphorylation, and degradation. To further clarify the role of Orc1A phosphorylation, we generated an antibody against phospho-peptides containing Ser^<276> and Orc1A protein is predominantly phosphorylated during G2/M phase. In addition, small amount of phosphorylated Orc1A gradually increases during S phase, while most of Orc1A remains unphosphorylated and bound to chromatin. All of the phosphorylated Orc1A is extracted in chromatin-unbound fraction containing 100 mM NaC1, Transiently expressed Orc1A is partially phosphoryla … More ted in the nucleus while nuclear translocation-deficient Orc1 mutant is extensively phosphorylated, We found that phosphorylation of Ser^<276> is dependent on conserved cyclin-docking (Cy) motif located just beside amino-terminal bromo adjacent homology (BAH) domain. Orc1 BAH domain has been shown to act as a binding site with Sir1 in S. cerevisiae or HP1 in Drosophila and Human. Taken together, we conclude that mouse Orc1 is regulated at least by phosphorylation of Ser^<276> residue throughout the cell cycle. At G1 phase, unphosphorylated Orc1A bound to chromatin and localized mainly on heterochromatin. During S phase, Orc1A is gradually released from chromatin, and released Orc1A is phosphorylated by CDK/cyclin A through Cy motif. Phosphorylated Orc1 is degraded during S phase. In contrast, at G2/M phase, phsophorylated Orc1 is not degraded but re-associates with heterochromatin after dephosphorylation. We are currently trying to understand whether Cy motif is masked by amino-terminal BAH domain on chromatin through interaction with HP1 or other chromatin factors. Less