Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts*
Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts*
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DOI：
10.1074/jbc.m600299200
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发表时间：
2006-04
影响因子：
4.8
通讯作者：
S. Waga;Akiko Zembutsu
中科院分区：
文献类型：
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作者：
S. Waga;Akiko Zembutsu
我们研究了非洲爪哇卵提取物中复制前复合体(Pre-RC)在质粒上形成过程中复制起始蛋白DNA结合的动态变化。在23℃的温度下，Pre-RC可以有效地形成在质粒上，每个质粒上都加载了一个或几个起源识别复合体(ORc)分子和∼10-20小染色体维护2(MCM2)分子。即使在双黄素存在的情况下(每粒含∼10个MCM2分子)，双黄素仍可抑制MCM的载量，但与质粒的相互作用很弱，但以化学计量学的方式依赖于ORC。有趣的是，在双黄素存在下，ORC、CDC6和CDT1DNA结合显著稳定，在这种条件下，ORC和CDC6各有∼10-20分子结合。此外，在较低的温度(0℃)下，不含人参皂苷的情况下，ORC-CDC_6-CD_(1 1)能迅速形成稳定的∼-CDC_6-CDT_1复合体，其中ORC和CDC_6分子各有10-20个分子与其结合，但几乎不与其结合。然而，当将温度改变到23℃时，大多数orc、cdc6和cdt1分子从DNA中移位，在质粒上留下大约一个orc分子，而∼10mcm2分子被加载到每个质粒上。此外，当分离的ORC-CDC6-CDT1-DNA复合体与纯化的MCM蛋白混合时，有可能将MCM负载到DNA上。这些结果表明，DNA上预先形成的ORC-CDC6-CDT1复合体直接参与了MCM的负载，并暗示在后生动物前RC形成过程中，每个DNA结合的ORC分子只负载一个或几个MCM2-7复合体。
We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23 °C, with one or a few origin recognition complex (ORC) molecules and ∼10–20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with ∼10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions ∼10–20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0 °C) without geminin, with ∼10–20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23 °C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas ∼10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2–7 complexes during metazoan pre-RC formation.