Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsBased 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.
Highlights
The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA
Given the fact that the zinc finger motifs were required for head-to-head double-hexamerization of the MtMCM [18], the 12-mers we identified here are not likely to be the head-to-head double hexamers
We found that most expressed nFL fragments of Mcm4, 6 and 7 formed aggregates or and did not behave well in solution
Summary
The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 forms a hexameric complex with helicase activity in vitro. Mcm functions as the replicative helicase, and can form various oligomeric complexes, including double-hexamers [5,6], hexamers [7,8], tetramers [9], trimers [10], and dimers [7,11,12]. It has been well demonstrated that Mcm are vital in the initiation and the elongation of genomic DNA replication as a eukaryotic replicative helicase. Helicase activity has been shown in vitro for MCM sub-complex comprising only three of the six subunits, Mcm4/6/7 hexamers (two copies of each subunit)
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