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BMC Biochemistry

, 14:5

Protein and enzyme biochemistry

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.

KeywordsCell cycle proteins DNA-binding proteins Recombinant proteins Protein binding Protein oligomerization Schizosaccharomyces pombe Escherichiai coli AbbreviationsMcmMinichromosome maintenance

S. pombeSchizosaccharomyces pombe

S. cerevisiaeSaccharomyces cerevisiae

pre-RCPre-replicative complex

ORCOrigin recognition complex

MtMethanothermobacter thermautotrophicus

SsoSulfolobus solfataricus

LTagSimian virus 40 large tumor antigen

E. coliEscherichiai coli

PCRPolymerase chain reaction

GSTGlutathione S-transferase

spS. pombe

scS. cerevisiae

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2091-14-5 contains supplementary material, which is available to authorized users.

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Autor: Meng Xu - Y Paul Chang - Xiaojiang S Chen

Fuente: https://link.springer.com/



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