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Open Access Highly Accessed Research article

Expression, purification and biochemical characterization of Schizosaccharomyces pombe Mcm4, 6 and 7

Meng Xu1, Y Paul Chang2 and Xiaojiang S Chen123*

Author affiliations

1 Graduate Program in Genetics, Molecular and Cell Biology, University of Southern California, Los Angeles, CA, 90089, USA

2 Molecular and Computational Biology Program, Departments of Biological Sciences/Chemistry, University of Southern California, Los Angeles, CA, 90089, USA

3 Norris Cancer Center, University of Southern California, Los Angeles, CA, 90089, USA

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Citation and License

BMC Biochemistry 2013, 14:5  doi:10.1186/1471-2091-14-5

Published: 27 February 2013

Abstract

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.

Keywords:
Cell cycle proteins; DNA-binding proteins; Recombinant proteins; Protein binding; Protein oligomerization; Schizosaccharomyces pombe; Escherichiai coli