Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex.
ABSTRACT Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.
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ABSTRACT: It has been reported that a point mutation of minichromosome maintenance (MCM)4 causes mammary carcinoma, and it deregulates DNA replication to produce abnormal chromosome structures. To understand the effect of this mutation at level of MCM2-7 interaction, we examined the effect of the same mutation of human MCM4 on the complex formation with MCM6 and MCM7 in insect cells. Human MCM4/6/7 complexes containing the mutated MCM4 were formed, but the hexameric complex formation was not evident in comparison with those containing wild-type MCM4. In binary expression of MCM4 and MCM6, decreased levels of MCM6 were recovered with the mutated MCM4, compared with wild-type MCM4. These results suggest that this mutation of MCM4 perturbs proper interaction with MCM6 to affect complex formation of MCM4/6/7 that is a core structure of MCM2-7 complex. Consistent with this notion, nuclear localization and MCM complex formation of forcedly expressed MCM4 in human cells are affected by this mutation. Thus, the defect of this mutant MCM4 in interacting with MCM6 may generate a decreased level of chromatin binding of MCM2-7 complex.Journal of biochemistry 06/2012; 152(2):191-8. · 1.95 Impact Factor
- 09/2011; , ISBN: 978-953-307-649-2
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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.BMC Biochemistry 02/2013; 14(1):5. · 1.78 Impact Factor