Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe
Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe
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DOI：
10.1074/jbc.m001118200
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发表时间：
2000-06-23
影响因子：
4.8
通讯作者：
Hurwitz, J
中科院分区：
文献类型：
--
作者：
Lee, JK;Hurwitz, J
微型染色体维持 (Mcm) 蛋白 2-7 在真核生物中高度保守，在 DNA 复制中发挥重要作用。在这里，我们描述了使用杆状病毒表达系统重建粟酒裂殖酵母Mcm蛋白的各种复合物。所有六种 Mcm 蛋白以及这些蛋白的不同组合的同时表达，产生了几种稳定的复合物，包括 Mcm2/3/4/5/6/7 的异六聚体、Mcm2/4/6/7 异四聚体、Mcm4/6/7 异三聚体的二聚体和 Mcm3/5 异二聚体。这些复合物的生化特性的纯化和表征表明，只有Mcm4/6/7 异源三聚体具有单链 DNA 依赖性 ATP 酶、ATP 依赖性单链 DNA 结合和 3' 至 5' DNA 解旋酶活性。与这些结果一致，Mcm2或Mcm3/5与Mcm4/6/7复合物的相互作用导致Mcm4/6/7二聚体复合物的分解以及DNA解旋酶活性的丧失。这些结果表明Mcm4/6/7复合物是Mcm复合物的催化核心，Mcm2和Mcm3/5可能参与该复合物活性的调节。
Minichromosome maintenance (Mcm) proteins 2-7 are highly conserved in eukaryotes and play an essential role in DNA replication. Here, we describe the reconstitution of the various complexes of the Mcm proteins of Schizosaccharomyces pombe using the baculovirus expression system. The simultaneous expression of all six of the Mcm proteins, as well as different combinations of these proteins, yielded several stable complexes that included the heterohexamer of Mcm2/3/4/5/6/7, the Mcm2/4/6/7 heterotetramer, the dimer of the Mcm4/6/7 heterotrimer, and the Mcm3/5 heterodimer, The purification and characterization of the biochemical properties of these complexes showed that only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities. Consistent with these results, the interaction of either Mcm2 or Mcm3/5 with the Mcm4/6/7 complex resulted in the disassembly of the dimeric complex of Mcm4/6/7 and the loss of DNA helicase activity. These results suggest that the Mcm4/6/7 complex is a catalytic core of the Mcm complex and that Mcm2 and Mcm3/5 may be involved in the regulation of the activity of this complex.