BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: In the fission yeastSchizosaccharomyces 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. RESULTS: 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. CONCLUSIONS: 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.
Authors: Xiong Yu; Margaret S VanLoock; Andrzej Poplawski; Zvi Kelman; Tao Xiang; Bik K Tye; Edward H Egelman Journal: EMBO Rep Date: 2002-07-15 Impact factor: 8.807