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Updated: May 13, 2026
Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast Schizosaccharomyces pombe
Published on: May 2, 2025
Expression, purification and biochemical characterization of Schizosaccharomyces pombe Mcm4, 6 and 7
Meng Xu1, Y Paul Chang, Xiaojiang S Chen
1Graduate Program in Genetics, Molecular and Cell Biology, University of Southern California, Los Angeles, CA 90089, USA.
Background:
The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 also forms a hexameric complex with helicase activity in vitro.
Results:
We used an Escherichiai coli expression system to express various domains of Schizosaccharomyces pombe Mcm4, 6 and 7 in order to characterize their domain structure, oligomeric states, and possible inter-/intra-subunit interactions. We also successfully employed a co-expression system to express Mcm4/6/7 at the same time in Escherichiai coli, and have purified functional Mcm4/6/7 complex in a hexameric state in high yield and purity, providing a means for generating large quantity of proteins for future structural and biochemical studies.
Conclusions:
Based on our results and those of others, models were proposed for the subunit arrangement and architecture of both the Mcm4/6/7 hexamer and the Mcm2-7 double-hexamer.
Insights
Researchers purified the Mcm4/6/7 complex, a key component of DNA replication machinery. This provides a reliable source for studying the minichromosome maintenance (MCM) protein complex structure and function.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Eukaryotic DNA replication relies on the minichromosome maintenance (MCM) protein complex.
- The MCM complex, a hetero-hexamer of Mcm2-7 proteins, exhibits helicase activity and forms double-hexamers on DNA.
- The Mcm4/6/7 subcomplex also possesses in vitro helicase activity.
Purpose of the Study:
- To characterize the domain structure, oligomeric states, and subunit interactions of Schizosaccharomyces pombe Mcm4, 6, and 7.
- To develop a method for producing functional Mcm4/6/7 hexameric complexes in high yield and purity.
Main Methods:
- Utilized an Escherichia coli expression system to express individual MCM protein domains.
- Employed a co-expression system in E. coli for simultaneous expression of Mcm4, 6, and 7.
- Purified the functional Mcm4/6/7 hexameric complex.
Main Results:
- Successfully expressed and characterized various domains of S. pombe Mcm4, 6, and 7.
- Developed a co-expression strategy to produce the Mcm4/6/7 complex.
- Purified the Mcm4/6/7 hexamer in high yield and purity, suitable for further studies.
Conclusions:
- The study provides a scalable method for obtaining functional Mcm4/6/7 hexamers.
- The results facilitate future structural and biochemical investigations of the MCM complex.
- Models for the subunit arrangement of Mcm4/6/7 and Mcm2-7 complexes were proposed.
