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

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.

Complejo mcm2-7. Revisión bibliográfica

“…However, contrary to this author, more recent genetic and biochemical studies show that the MCM2-7 complex subunits have different functions (16)(17)(18) . As mentioned above, helicase activity has been demonstrated in vitro for the MCM4, 6 and 7 subcomplex (12) , while MCM2, 3 and 5 are responsible for activating and deactivating the complex (10)(11) . Given the high activity of the MCM2-7 present in the cells during the cell cycle, and the fact that these proteins are absent in quiescent cells, the different proteins in the MCM2-7 complex could be good markers of cell proliferation (19) , possible biological markers for diagnosis and prognosis (20)(21) and future therapeutic targets (22) .…”