In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric (Ϸ10 3 kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3-to-5 ATPdependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.replication ͉ ORC1 protein ͉ Cdc6 protein T he minichromosome maintenance (MCM) genes encode a family of proteins first identified by their essential role in the maintenance of ARS-containing minichromosomes in Saccharomyces cerevisiae (1). MCM homologues have been identified in all eukaryotes from yeast to mammals and have highly conserved amino acid sequences (2, 3). The MCM family consists of at least six distinct polypeptides (MCM2-7). In addition to forming a heterohexameric structure, the MCM proteins are capable of forming various subcomplexes in vitro (4-8). Although the MCM proteins were shown to play an important role in the initiation and elongation phases of DNA replication, the precise functions of the MCM complexes are not yet fully understood (reviewed in refs. 3, 9, and 10). In vitro studies have revealed several biochemical properties of the MCM proteins. A heterotrimeric complex of human (h) MCMs (MCM4, -6, and -7) that forms a dimeric structure contains ATP-dependent DNA helicase activity, binds to single-stranded DNA, and possesses DNA-dependent ATPase activity (11). It was also demonstrated that the hMCM2 protein interacts with the hMCM4, -6, and -7 complex to form the heterotetrameric complex, hMCM2, -4, -6, and -7, which is devoid of the activities associated with the hMCM4, -6, and -7 complex (11, 12). Identical observations have been made with MCM proteins isolated from Schizosaccharomyces pombe (spMCM; J.-K.L. and J.H., unpublished observations).In addition to genetic studies that demonstrated the essential role of the MCM proteins in DNA replication, biochemical studies showed that these proteins are assembled onto origins of replication in S. cerevisiae (13,14). Furthermore, the proteins appear to interact genetically and physically with the origin recognition complex (ORC) and other proteins that participate in the initiation of DNA replication such as . These genetic data obtained from yeast, together with the in vitro biochemical properties, have led to the suggestion that the MCM proteins may function as the replicative helicase in eukaryotes, similar to role of the bacterial DnaB protein in Escherichia coli replication and the large tumor antigen (T Ag) in the replication of simian virus 40 (SV40) .Archaea, the third domain of life (21), are believed to replicate DNA in a eukaryotic-like f...

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In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G1/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.T he initiation of eukaryotic DNA replication is a multistep process that requires the assembly of the prereplicative complex (pre-RC), activation of the pre-RC and formation of the replisome (1, 2). Pre-RC assembly occurs during the G 1 phase of the cell cycle by a stepwise recruitment of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex onto DNA origins. During the G 1 /S transition stage, the recruitment of other replication factors such as Cdc45 and GINS and the combined actions of two S phase promoting kinases, the cyclindependent (CDK) and Cdc7-Dbf4 (DDK) kinases, lead to the assembly of an active DNA helicase complex at replication origins. This activation results in the unwinding of replication origins, the recruitment of DNA polymerases and accessory factors and the assembly of the replisome for DNA synthesis.In eukaryotic cells, the Mcm2-7 complex seems to be the catalytic core of the replicative helicase. It is essential for origin unwinding and replication fork progression (3-6). It has been shown that a variety of Mcm complexes, including the Mcm4/6/7 subcomplex (from many species), double hexameric complexes of Mcm homologues in Archaea and the budding yeast Mcm2-7 complex, exhibit DNA helicase activity in vitro (1, 7). In vivo, however, formation of an active helicase at replication origins requires the further recruitment of several factors including Cdc45 and GINS, and this activation process is governed by CDK and DDK (1).Both Cdc45 and GINS are required for the establishment and progression of the replication fork (8, 9). These proteins are loaded onto origins during S phase and form a stable complex with Mcm2-7 (10). The complex seems to be a DNA unwinding complex and moves along DNA as part of the replication fork complex (6, 11). Consistent with these observations, a complex of Mcm2-7, Cdc45, and GINS (the CMG complex), purified from D...

Cdc7 is an essential kinase required for the initiation of eukaryotic DNA replication. Previous studies in many species showed that the minichromosome maintenance complex is a major physiological target of this kinase. In this study, we have mapped the sites in human Mcm2 protein that are phosphorylated by Cdc7. The in vitro phosphorylation of several Mcm2 truncated proteins and peptides revealed that Mcm2 contains two major ( 5 S and 53 S) and at least three minor phosphorylation sites ( 4 S, 7 S, and 59 T) located at the N-terminal region. Alanine substitution experiments with Mcm2 peptides showed that the phosphorylation of 5 S and 53 S by Cdc7 required the presence of an acidic amino acid adjacent to a serine residue. Furthermore, although Cdc7 was unable to phosphorylate a Mcm2 peptide (spanning amino acids 19 -30 and containing 26 S and 27 S), it phosphorylated 26 S efficiently when this peptide contained a chemically synthesized phospho-27 S modification. Hence, additional Cdc7 phosphorylation sites could be generated in Mcm2 by its prior phosphorylation by a cyclin-dependent kinase. This finding may explain why the sequential action of cyclindependent and Cdc7 kinases is essential for the initiation of DNA replication.replication ͉ prereplicative complex ͉ origin activation C dc7 plays an essential role in the initiation of eukaryotic DNA replication (1-3). Cdc7 encodes a serine͞threonine kinase that is highly conserved from yeast to human (4). The activity of this kinase fluctuates during the cell cycle and depends completely on the regulatory subunit, Dbf4. Dbf4 accumulates during the S and G 2 phases of the cell cycle and is degraded rapidly during the G 1 phase by the anaphase promoting complex (5, 6). In Saccharomyces cerevisiae, Dbf4 binds to chromatin at the G 1 ͞S transition and remains on chromatin during the S phase (7). In the Xenopus egg extract cell-free system, Cdc7 was found to bind to chromatin during S phase, and this association required the minichromosome maintenance (MCM) complex (8).Both genetic and biochemical studies in S. cerevisiae indicate that Cdc7 kinase activity is required throughout S phase to activate replication origins on chromosomes (9). Cdc7 kinase acts directly on individual replication origins, presumably by phosphorylating components of the prereplicative complex (pre-RC), which may lead to the remodeling of the pre-RC for the unwinding of replication origins and subsequent recruitment of the replication fork machinery. Although the mechanism-oforigin activation by Cdc7 kinase remains unclear, the MCM complex appears to be the primary physiological target of this kinase activity (2).The MCM complex is composed of six distinct subunits (Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, and Mcm7), which are all related structurally and highly conserved in eukaryotes (10). All six proteins are essential for the initiation of DNA replication and contain sequence motifs required for DNA helicase activity. In keeping with this notion, the MCM subcomplex containing Mcm4, Mcm6, and Mcm7 posses...

The interaction between an origin sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication. In this report, we have examined the interaction between the Schizosaccharomyces pombe (sp) autonomously replicating sequence 1 (ars1) and the spORC. For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminaldeleted spOrc4 subunit (spORC ), and the spOrc4 subunit by using the baculovirus expression system. Wild-type spORC showed sequence-specific binding to ars1, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC. In contrast, the spORC ⌬N-Orc4 and the ⌬N-spOrc4p alone did not bind significantly to ars1. These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the ars1-binding activity. DNA-binding competition assays with fragments of ars1 and DNase I footprinting studies with full-length ars1 revealed that the spORC interacted with several AT-rich sequence regions of ars1. These DNA-binding properties of spORC correlate with the previously determined sequence requirements of the S. pombe ars1. These studies indicate that because of its unique Orc4 subunit, S. pombe uses a mechanism to recognize its origins different from that used by Saccharomyces cerevisiae. D NA replication origin sequences in Saccharomyces cerevisiaeare relatively short AT-rich sequences (100-150 bp) that include an essential autonomously replicating sequence (ARS) consensus sequence (ACS) (1-3). In contrast, replicators in higher eukaryotes are much longer and share no recognizable essential motifs (4, 5). The ARS elements of Schizosaccharomyces pombe are larger (longer than 700 bp) than those of Sac. cerevisiae, and contain several redundant AT-rich sequences that are important for ARS activity, but lack a conserved essential sequence motif such as an ACS (6-8).The origin recognition complex (ORC) was initially purified as an ARS-binding protein from Sac. cerevisiae (9). ORC consists of six distinct subunits, Orc1p to Orc6p, which are all essential for DNA replication (10-12). Sac. cerevisiae ORC specifically recognizes and binds to the ACS containing an 11-bp AT-rich sequence in vivo and in vitro, and ATP is essential for this ARS-binding activity (9,13,14). ORC binds to replication origin DNA throughout the cell cycle and is required to recruit other essential initiation factors for the assembly of the prereplicative complex (15,16). ORC is conserved in all eukaryotes and has been identified and purified from other species, including S. pombe, Drosophila melanogaster, Xenopus laevis, and humans (17)(18)(19)(20)(21)(22). Although in vivo and in vitro studies suggest that ORC is essential for DNA replication in these species (15,16), the interactions between these ORCs and their replication origin DNAs have not been characterized except for Sac. cerevisiae. D. melanogaster ORC interacts with multip...