Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex. J Biol Chem
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.
- SourceAvailable from: Lori Frappier
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- "We also found that a lysine-to-alanine point mutation within the conserved Walker A motif of MCM7 did not impair this interaction (Supplemental Fig. S4B). Given that the lysine residue in the Walker A motif is essential for nucleotide-binding and DNA-unwinding activities (You et al. 2002), this result suggests that ATP or its hydrolysis is not necessary for MCM7 to be associated with MCM-BP. MCM-BP is imported in the nucleus in late S phase, when MCM2–7 dissociates from chromatin The association of MCM-BP with MCM7, a subunit of the MCM2–7 helicase, led us to ask whether MCM-BP could play a role during DNA replication. "
ABSTRACT: Origins of DNA replication are licensed by recruiting MCM2-7 to assemble the prereplicative complex (pre-RC). How MCM2-7 is inactivated or removed from chromatin at the end of S phase is still unclear. Here, we show that MCM-BP can disassemble the MCM2-7 complex and might function as an unloader of MCM2-7 from chromatin. In Xenopus egg extracts, MCM-BP exists in a stable complex with MCM7, but is not associated with the MCM2-7 hexameric complex. MCM-BP accumulates in nuclei in late S phase, well after the loading of MCM2-7 onto chromatin. MCM-BP immunodepletion in Xenopus egg extracts inhibits replication-dependent MCM dissociation without affecting pre-RC formation and DNA replication. When excess MCM-BP is incubated with Xenopus egg extracts or immunopurified MCM2-7, it binds to MCM proteins and promotes disassembly of the MCM2-7 complex. Recombinant MCM-BP also releases MCM2-7 from isolated late-S-phase chromatin, but this activity is abolished when DNA replication is blocked. MCM-BP silencing in human cells also delays MCM dissociation in late S phase. We propose that MCM-BP plays a key role in the mechanism by which pre-RC is cleared from replicated DNA in vertebrate cells.Genes & development 01/2011; 25(2):165-75. DOI:10.1101/gad.614411 · 12.64 Impact Factor
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- "The contribution that each ATP site makes to the activity of the complex appears to be roughly equal (about 35-50% of wild type ATPase activity and all interfered with DNA unwinding). This observation contrasts to the studies of You and colleagues on mouse Mcm4/6/7, which indicated that each ATP site made different contributions to ATP hydrolysis, DNA binding and DNA unwinding . The mouse study used mutations in the Walker A and B motifs, which in some proteins affects ATP binding as well as hydrolysis and this may account for the differences. "
ABSTRACT: Minichromosome maintenance proteins (Mcm) 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7. Here, we show that Saccharomyces cerevisiae Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants) indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer. The minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.BMC Biochemistry 09/2010; 11(1):37. DOI:10.1186/1471-2091-11-37 · 1.94 Impact Factor
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- "To more directly examine whether DNA unwinding at relicensed origins was required for generating ssDNA, we inhibited MCM helicase activity by overexpressing an MCM7 mutant (K387A) carrying a mutation at its conserved ATPase motif that is required for replication helicase activity (You et al., 1999; You et al., 2002). Immunoprecipitation showed that MCM7-K387A interacted with other endogenous MCM proteins (not depicted) and that overexpression of this mutant prevented cells from entering S phase, suggesting a dominant-negative effect of this mutant (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200704138/DC1). "
ABSTRACT: DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)-mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.The Journal of Cell Biology 12/2007; 179(4):643-57. DOI:10.1083/jcb.200704138 · 9.69 Impact Factor