Adachi Y, Usukura J, Yanagida M
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Genes Cells. 1997 Jul;2(7):467-79. doi: 10.1046/j.1365-2443.1997.1350333.x.
In the fission yeast Schizosaccharomyces pombe, Nda1, Nda4, Mis5 and Cdc21 proteins belong to the MCM (minichromosome maintenance) protein family which is thought to have six members. Each MCM member is required for the early stages of DNA replication, and has a well-conserved central 200-amino acid domain containing a putative ATP binding motif. However, the precise molecular functions of MCM proteins are not yet clear.
We investigated the physical interaction of Nda1 protein with the other fission yeast MCM proteins using specific antibodies. Immunoprecipitation of Nda1 protein leads to the co-precipitation of all the other members of the fission yeast MCM protein family. We purified the MCM protein complex by a combination of column chromatography. The native molecular weight of the MCM complex was estimated by gel filtration to be 560 kDa. The purified fraction contained nearly equal quantities of the six MCM proteins. Electron microscope observation showed that the MCM complex has a globular shape with a central cavity.
We have developed a procedure to purify fission yeast MCM proteins in a native hetero-oligomeric complex form for the first time, which opens an avenue to further biochemical analysis.
在裂殖酵母粟酒裂殖酵母中，Nda1、Nda4、Mis5和Cdc21蛋白属于MCM（微小染色体维持）蛋白家族，该家族被认为有六个成员。每个MCM成员在DNA复制的早期阶段都是必需的，并且有一个保守的中央200个氨基酸的结构域，其中包含一个假定的ATP结合基序。然而，MCM蛋白的确切分子功能尚不清楚。
我们使用特异性抗体研究了Nda1蛋白与其他裂殖酵母MCM蛋白之间的物理相互作用。Nda1蛋白的免疫沉淀导致裂殖酵母MCM蛋白家族的所有其他成员共沉淀。我们通过柱色谱法的组合纯化了MCM蛋白复合物。通过凝胶过滤估计MCM复合物的天然分子量为560 kDa。纯化的组分含有几乎等量的六种MCM蛋白。电子显微镜观察表明，MCM复合物呈球形，有一个中央腔。
我们首次开发了一种以天然异源寡聚体复合物形式纯化裂殖酵母MCM蛋白的方法，这为进一步的生化分析开辟了一条途径。
