Biochemical Characterization of the Minichromosome Maintenance (MCM) Protein of the Crenarchaeote Aeropyrum pernix and Its Interactions with the Origin Recognition Complex (ORC) Proteins
Biochemical Characterization of the Minichromosome Maintenance (MCM) Protein of the Crenarchaeote Aeropyrum pernix and Its Interactions with the Origin Recognition Complex (ORC) Proteins
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DOI：
10.1021/bi801479s
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发表时间：
2008-12-16
期刊：
影响因子：
2.9
通讯作者：
Grainge, Ian
中科院分区：
文献类型：
--
作者：
Atanassova, Neli;Grainge, Ian
古细菌中的复制是由真核生物的同源蛋白质进行的。然而，古细菌系统往往比真核生物系统简单得多，编码核心功能的不同基因更少。在许多古细菌中，有一个单一的微型染色体维持（MCM）同源物，推测是复制解旋酶和一个和三个之间的起源识别复合物（ORC）的同源物参与结合到复制起点。在这里，我们描述的MCM蛋白质的克隆和表征从crenarchaeote Aeropyrum pernix。与其他真核生物和古细菌MCM蛋白一样，它被发现是一种ATP依赖性DNA解旋酶，并且通过突变证实了参与ATP结合和水解的推定活性位点残基。N-末端256个氨基酸的缺失产生了在不存在DNA的情况下具有更高ATP酶活性的蛋白质，并保留了稳健的解旋酶活性。与A. pernix的解旋酶活性，发现两种ORC同系物都能抑制MCM的解旋酶活性。进一步发现ORC 2在ATP存在下可以自磷酸化，并且更显著地可以以种特异性方式磷酸化MCM。
Replication in archaea is carried out by proteins that are homologues of eukaryotic counterparts. However, the archaeal systems tend to be much simpler with fewer different genes encoding the core functions than in eukaryotic counterparts. In many archaea, there is a single minichromosome maintenance (MCM) homologue, presumed to be the replicative helicase and between one and three origin recognition complex (ORC) homologues involved in binding to the replication origins. Here we describe the cloning and characterization of the MCM protein from the crenarchaeote Aeropyrum pernix. Like other eukaryotic and archaeal MCM proteins, it is found to be an ATP-dependent DNA helicase, and the putative active site residues involved in ATP binding and hydrolysis are confirmed by mutation. Deletion of the N-terminal 256 amino acids yielded a protein with higher ATPase activity in the absence of DNA and retained robust helicase activity. Interactions with the ORC proteins of A. pernix were examined, and it was found that both ORC homologues could inhibit the helicase activity of MCM. Further it was found that ORC2 could autophosphorylate in the presence of ATP and more remarkably could phosphorylate MCM in a species-specific manner.