Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast

The Cdc6 protein is essential for the assembly of pre-replicative complexes (pre-RCs) at origins of DNA replication in the budding yeast Saccharomyces cerevisiae. This reaction is blocked in vivo by the cyclin-dependent kinase Cdc28p, together with its regulatory subunits, the B type cyclins that are present throughout S, G₂, and M phases. Because the destruction of B type cyclins and the consequent inactivation of the kinase are essential for exit from mitosis, pre-RC formation can only occur after passage through mitosis. Therefore, pre-RC formation has been proposed to be essential for coupling S phase and mitosis and for limiting DNA replication to once per cell cycle. The Mcm2–7 family of proteins has been implicated in limiting replication to once per cell cycle from experiments with Xenopus egg extracts. Here we show that the Mcm proteins of budding yeast are abundant and are quantitatively found in a chromatin-enriched fraction specifically during the G₁ phase of the cell cycle. This chromatin binding depends on the de novo synthesis of Cdc6p, providing evidence that a conserved biochemical pathway plays a critical role in coordinating DNA replication with mitosis in both yeast and higher eukaryotes. Cdc6p and the origin recognition complex can be selectively removed from this chromatin-enriched fraction without removing the Mcm proteins. From these results, we propose that Cdc6p (and the origin recognition complex) nucleates the binding of Mcm proteins to chromatin, but once bound, the Mcm proteins appear to interact tightly with some other component of chromatin.     

CDC45 is required in conjunction with CDC7/DBF4 to trigger the initiation of DNA replication

The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene. We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G₁, after START and prior to DNA synthesis. Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins. These complexes are then triggered at the onset of S phase to commence DNA replication. Cells defective for CDC45 are incapable of activating the complexes to initiate DNA replication. In addition, Cdc45p and Cdc7p/Dbf4p, a kinase implicated in the G₁/S phase transition, are dependent on one another for function. These data indicate that CDC45 functions in late G₁ phase in concert with CDC7/DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes.     

Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.

We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect. Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins. We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p. These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes. Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1.     

Identification and characterization of a human protein kinase related to budding yeast Cdc7p

The Cdc7p protein kinase is essential for the G₁/S transition and initiation of DNA replication during the cell division cycle in Saccharomyces cerevisiae. Cdc7p appears to be an evolutionarily conserved protein, since a homolog Hsk1 has been isolated from Schizosaccharomyces pombe. Here, we report the isolation of a human cDNA, HsCdc7, whose product is closely related in sequence to Cdc7p and Hsk1. The HsCdc7 cDNA encodes a protein of 574 amino acids with predicted size of 64 kDa. HsCdc7 contains the conserved subdomains common to all protein-serine/threonine kinases and three “kinase inserts” that are characteristic of Cdc7p and Hsk1. Immune complexes of HsCdc7 from cell lysates were able to phosphorylate histone H1 in vitro. Indirect immunofluorescence staining demonstrated that HsCdc7 protein was predominantly localized in the nucleus. Although the expression levels of HsCdc7 appeared to be constant throughout the cell cycle, the protein kinase activity of HsCdc7 increased during S phase of the cell cycle at approximately the same time as that of Cdk2. These results, together with the functions of Cdc7p in yeast, suggest that HsCdc7 may phosphorylate critical substrate(s) that regulate the G₁/S phase transition and/or DNA replication in mammalian cells.     

Assembly of the Cdc45-Mcm2–7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins

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.     

CDC7 kinase phosphorylates serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein

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 cyclin-dependent and Cdc7 kinases is essential for the initiation of DNA replication.     

Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks

The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2–7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation.     

Interaction of the S phase regulator Cdc18 with cyclin-dependent kinase in fission yeast

The fission yeast gene cdc18⁺ is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G₁/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo. We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2⁺ gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G₁/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.     

Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.Eukaryotic DNA replication initiates from multiple replication origins within each chromosome to duplicate the large genome efficiently. To ensure DNA synthesis occurs once and only once across the genome, cells adopt a two-step process to activate replication origins during two separate stages of the cell-division cycle. The first step is licensing of replication origins, which occurs only when cyclin-dependent kinase (CDK) activity is low. In Saccharomyces cerevisiae, origins of DNA replication are licensed in G1 by the formation of a prereplicative complex (pre-RC). The process begins with the origin recognition complex binding to replication origins and recruiting the licensing factor Cdc6, which facilitates loading of the Cdt1-bound minichromosome maintenance (MCM) complex composed of Mcm2–Mcm7 (Mcm2–7). The hexameric Mcm2–7 is the core of the replicative helicase that unwinds DNA during replication. Within the pre-RC Mcm2–7 is loaded as an inactive double hexamer. The next step, activation of licensed origins (origin firing), occurs throughout the S phase and requires the continuous presence of two kinases, the S phase CDKs and the Dbf4-dependent Cdc7 kinase (DDK). CDK phosphorylates Sld2 and Sld3 to allow their binding to Dpb11 (1, 2), facilitating recruitment of Cdc45 and GINS (composed of protein subunits Sld5, Psf1, Psf2 and Psf3; Go, Ichi, Nii, and San stand for five, one, two, and three in Japanese, respectively) to Mcm2–7 to create an active helicase. DDK phosphorylates Mcm2–7 and blocks an intrinsic initiation inhibitory activity residing in the N terminus of the Mcm4 subunit (3). The concerted action of these S-phase kinases transforms the inactive Mcm2–7 double hexamer into the active helicase complex composed of Cdc45, Mcm2-7, and GINS (the CMG complex) (4–6). Upon initiation, DNA polymerases and other components of the replication machinery are recruited to form replisomes and establish replication forks, where DNA synthesis ensues.Kinase-signaling pathways target various components of the replication machinery. Both CDK and DDK target replication proteins in addition to their essential targets described above. Furthermore, Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related (ATM/ATR) signaling targets components of the CMG helicase complex under replication stress (7–10). In the yeast S. cerevisiae, DNA damage activates the checkpoint kinase Rad53, which phosphorylates both Sld3 and Dbf4 to inhibit late origin firing (11, 12). The yeast ATM/ATR homolog Mec1 also targets Mcm4 (13). The stress-activated protein kinase Hog1 targets an auxiliary replisome component Mrc1 to regulate both origin firing and fork progression (14). Although we now have a better understanding of the essential functions of protein kinases in controlling the initiation of replication, we do not completely understand how the separate kinase signaling pathways are coordinated to regulate both initiation and replication fork progression.The structurally disordered N-terminal serine/threonine-rich domain (NSD) of Mcm4 is a target of multiple kinases, including DDK, CDK, and Mec1 (3, 13, 15, 16). Within this region we have identified two functionally distinct domains that exert different functions and are regulated by different kinase systems even though they overlap extensively in primary amino acid sequences. The segment of the Mcm4 NSD proximal to the DDK-docking domain (DDD) (15), and hence termed “proximal NSD,” blocks initiation until it is phosphorylated by DDK. In contrast, the distal segment of the NSD at the N terminus, away from the DDD, is targeted by additional kinases and contributes positively to promote S-phase progression. In this study we present a comprehensive analysis of the pattern of origin activation, replication fork progression, and the checkpoint response in cells under replication stress caused by the inhibition of ribonucleotide reductase (RNR). We show that the distal and proximal NSD segments contribute differently to origin activation and DNA replication fork progression. Furthermore, they exert opposing effects on checkpoint signaling under replication stress. All these effects are regulated by phosphorylation. We suggest that the Mcm4 NSD, a regulatory domain intrinsic to the replicative helicase, mediates the control of multiple aspects of DNA replication. Our data reveal a sophisticated mechanism to fine-tune S-phase progression in response to changing environments.     

Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase

The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2-7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.     

Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase

Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase.The replication fork helicase in eukaryotes is composed of Cdc45, the Mcm2-7 heterohexameric ATPase, and the tetrameric GINS (Go, Ichi, Ni, and San) complex (CMG assembly) (1). The replication fork helicase (CMG) assembles in S phase in a manner that is dependent upon the replication initiation factors Sld2, Sld3, and Dpb11 (2). Sld3 (Treslin/TICRR in humans), Sld2 (RecQL4/RecQ4 in humans), and Dpb11 (TopBP1 in humans) are required for the initiation of DNA replication, but these proteins do not travel with the replication fork (3). The S-phase-specific kinases, cyclin-dependent kinase (CDK) and the Dbf4-dependent kinase (DDK), are also required for CMG assembly and origin activation (4, 5). In late M and G₁ phases, the Mcm2-7 complex loads to encircle dsDNA as a double hexamer (6, 7). During S phase, a single strand of DNA is extruded from the central channel of Mcm2-7, and this event is required because the CMG complex unwinds DNA by a steric exclusion mechanism (8).Central to the initiation of DNA replication is the coordination of entry into S phase with origin firing (4, 5). Levels of the S phase-specific kinases, S-CDK and DDK, rise during the onset of S phase, and these two kinases are central to coordinating S-phase entry with origin firing (4, 5). S-CDK phosphorylates Sld2 and Sld3, and these phosphorylation events are the essential functions of S-CDK (9, 10). S-CDK phosphorylation of Sld3 is conserved in human Treslin (11). S-CDK phosphorylation of Sld2 promotes the association of Sld2 with yeast Dpb11 (12), and also the association of Sld2 with T-rich ssDNA (13). S-CDK phosphorylation of Sld3 stimulates the association of Sld3 with Dpb11 (9, 10). The associations of Sld2 with Dpb11 and Sld3 with Dpb11 have been proposed to be important for the recruitment of GINS to origins, through the generation of a preloading complex (Pre-LC), composed of Sld2, GINS, Polε, and Dpb11 (14). S-CDK–catalyzed formation of an Sld3-Dpb11-Sld2 complex has also been proposed to be important to generate a ternary ssDNA-binding complex of high affinity, because Sld2, Sld3, and Dpb11 bind to T-rich ssDNA (13, 15, 16).The essential role of DDK in yeast cells is the phosphorylation of subunits of the Mcm2-7 complex (17). DDK phosphorylation of Mcm4 is important for cell growth, and this phosphorylation event alleviates an inhibitory function of the N terminus of Mcm4 (18). DDK phosphorylation of Mcm4 may also promote the interaction between Cdc45 and Mcm2-7 (18). DDK phosphorylation of Mcm6 may also be important for cell growth (19). Mcm2 is also a target for DDK (20), and DDK phosphorylation of Mcm2 is also required for DNA replication under normal growth conditions (21). Furthermore, expression of a mutant of mcm2 (mcm2-S164A,S170A) that is not phosphorylated by DDK exerts a dominant-negative severe growth defect in budding yeast that is bypassed by the mcm5-bob1 (mcm5-P83L) mutation (21). The biochemical mechanism of this genetic suppression has also been examined. DDK phosphorylation of Mcm2 reduces the affinity of budding yeast Mcm2 for Mcm5, and the mcm5-bob1 mutation also reduces this affinity (21). This reduced affinity may help open the “Mcm2-Mcm5 gate,” which may be important for the extrusion of ssDNA from the central channel of Mcm2-7 during S phase, a requirement for origin activation (22).Cdc45 binds weakly to Mcm2-7 in the absence of accessory factors (23). Sld3 binds tightly to Mcm2-7 and Cdc45, and thus Sld3 recruits Cdc45 to Mcm2-7 complexes (2, 23). This step may further require DDK and involve the nonessential initiation factor Sld7 (24). During origin activation, Sld3 is removed from Mcm2-7, presumably through the exposure of sequestering T-rich ssDNA (16). GINS can substitute for Sld3 as a factor that promotes the association of Cdc45 with Mcm2-7, thereby forming the stable Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex (23, 25).The mechanism of GINS recruitment may involve the formation of the S-CDK–dependent preloading complex, wherein the pre-LC recruits GINS to Mcm2-7, analogous to how Sld3 recruits Cdc45 to Mcm2-7 (14). A second proposal posits that Sld3, Sld2, and Dpb11 compete with GINS for binding to Mcm2-7 before origin activation, blocking the premature interaction between GINS and Mcm2-7 before origin activation (15, 16, 26). However, when T-rich ssDNA is extruded from the central channel of Mcm2-7, an ssDNA binding surface for Sld3-Sld2-Dpb11 is generated (15, 16, 26). Sld3-Sld2-Dpb11 dissociates from Mcm2-7 once the origin is melted, because Sld3-Sld2-Dpb11 has a higher affinity for ssDNA then Mcm2-7 (15, 16, 26). The dissociation of Sld3-Sld2-Dpb11 from Mcm2-7 allows GINS to bind Mcm2-7 by a passive, sequestration mechanism (15, 16, 26). The two models are not incompatible with one another, and they may both be correct.There is an excess of Mcm2-7 double hexamer complexes loaded onto dsDNA in M phase and G₁ relative to the number of Mcm2-7 double hexamer complexes that actually fire. Remarkably, the activated Mcm2-7 complexes share several features in common: DDK phosphorylation of Mcm2-7, initiation factor (Sld3, Sld2, Dpb11, Mcm10, and Sld7) binding to Mcm2-7, and Cdc45/GINS attachment to Mcm2-7. What coordinates these different activities at a particular Mcm2-7 double hexamer? In other words, what prevents Cdc45 from binding to one Mcm2-7 double hexamer while DDK phosphorylates a different Mcm2-7 double hexamer? We sought to address this fundamental question in this paper.We show that DDK-phosphorylated Mcm2 preferentially interacts with Cdc45 in vivo compared with Mcm2 (all Mcm2 in cell, phosphorylated and unphosphorylated), suggesting that Cdc45 recruitment to Mcm2-7 is correlated with DDK phosphorylation of Mcm2. We also show that Sld3 substantially stimulates DDK phosphorylation of Mcm2 in vitro. We identified a mutant of Sld3, sld3-m16, that is defective in the stimulation of DDK phosphorylation of Mcm2. When sld3-m16 is expressed in budding yeast cells, a dominant negative severe growth defect is observed that is bypassed by mcm5-bob1. Wild-type expression levels of sld3-m16 confer a growth and DNA replication defect, with decreased DDK phosphorylation of Mcm2. Furthermore, expression of sld3-m16 results in diminished replication protein A (RPA)-ChIP signal at an origin, and decreased GINS interaction with Mcm2-7. Furthermore, human Treslin substantially stimulates DDK phosphorylation of Mcm2 at serines 53 and 108. Finally, a mutant of human Mcm2 that mimics DDK-phosphorylated Mcm2 exhibits diminished interaction with human Mcm5, suggesting a mechanism for “gate” opening. We conclude that Sld3 coordinates helicase phosphorylation with helicase assembly.     

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.     

The Cdc6p nucleotide-binding motif is required for loading Mcm proteins onto chromatin

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin-associated factor. It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6^K114E protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.     

A human protein related to yeast Cdc6p

The unstable proteins Cdc6p and cdc18⁺ are essential and rate limiting for the initiation of DNA replication in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively, and also participate in checkpoint controls that ensure DNA replication is completed before mitosis is initiated. We have identified Xenopus and human proteins closely related to Cdc6p/cdc18. The human protein, p62^cdc6, is encoded on chromosome 17q21.3 and includes putative cyclin-dependent kinase phosphorylation sites, destruction boxes, a nucleotide binding/ATPase domain, and a potential leucine zipper. Expression of p62^cdc6 mRNA and protein is suppressed in human diploid fibroblasts made quiescent by serum starvation, and peaks as cells reenter the cell cycle and replicate DNA following serum stimulation. Conservation of structure among proteins involved in initiation suggests that fundamental features of replication complexes are maintained in all eukaryotes.     

The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1 kinase

Previous studies in Saccharomyces cerevisiae have defined an essential role for the Dbf4-Cdc7 kinase complex in the initiation of DNA replication presumably by phosphorylation of target proteins, such as the minichromosome maintenance (Mcm) complex. We have examined the phosphorylation of the Mcm complex by the Dfp1-Hsk1 kinase, the Schizosaccharomyces pombe homologue of Dbf4-Cdc7. In vitro, the purified Dfp1-Hsk1 kinase efficiently phosphorylated Mcm2p. In contrast, Mcm2p, present in the six-subunit Mcm complex, was a poor substrate of this kinase and required Cdc23p (homologue of Mcm10p) for efficient phosphorylation. In the presence of Cdc23p, Dfp1-Hsk1 phosphorylated the Mcm2p and Mcm4p subunits of the Mcm complex. Cdc23p interacted with both the Mcm complex and Dfp1-Hsk1 by selectively binding to the Mcm467 subunits and Dfp1p, respectively. The N terminus of Cdc23p was found to interact directly with Dfp1-Hsk1 and was essential for phosphorylation of the Mcm complex. Truncated derivatives of Cdc23p that complemented the temperature-sensitive phenotype of cdc23 mutant cells also stimulated the phosphorylation of Mcm complex, implying that this activity might be a critical role of Cdc23p in vivo. These results suggest that Cdc23p participates in the activation of prereplicative complex by recruiting the Dfp1-Hsk1 kinase and stimulating the phosphorylation of the Mcm complex.     

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2–7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2–7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2–7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel.Chromosomal DNA replication begins with the separation of the complementary strands of the duplex. Following this melting step, helicases continuously separate the paired strands, exposing the template for enzymatic synthesis. For eukaryotic DNA replication, a growing body of work suggests that the initial DNA melting step involves an enzymatic conversion of a double hexamer of the minichromosome maintenance (Mcm2–7) complex into an active helicase, with hexamer separation forming two forks moving in opposite directions (1). The molecular mechanisms and a complete list of the factors that work to achieve melting and the topological conversion of double to single strand are still unknown, but both Cdc45 and the GINS complex are present at the time of melting (2) and are critical components of an activated Mcm2–7 helicase (3–6). Understanding the initiation process requires studies focused on the various transitions accessed by Mcm2–7 proteins and defining what roles the GINS and Cdc45 may play in the melting and unwinding processes.The CMG helicase characterized in vitro in Drosophila and humans contains a single Cdc45 protein, a single hetero-hexameric Mcm2–7, and a tetrameric GINS complex (3–7). Mcm2–7 acts as the motor that drives helicase activity; however, for metazoans, only when the Mcm2–7 complex is associated with Cdc45 and GINS do significant helicase, ATPase, and DNA binding activities follow (4). The two Mcm2–7 complexes are first loaded to DNA by mechanisms that appear to retain certain parallels to the processes used for loading sliding processivity clamps onto DNA. The endpoint of loading results in the deposition of a duplex DNA that runs through the central channel of an MCM double hexamer. Although topologically linked to DNA, the associated Mcm2–7 double hexamer appears to only weakly engage the duplex, as it can slide off a linearized segment in the absence of a blocking barrier (8, 9).Following double-hexamer formation, a transition is thought to occur whereby the lagging strand exits the central channel of the MCM ring and the leading strand remains in the central channel. Evidence for this transition follows from biochemical studies showing that the replication fork can bypass a roadblock on the lagging (5′–3′) but not the leading (3′–5′) strand (10) and that the CMG can only bind single-stranded DNA presented at a fork (4). Similarly, both biochemical and structural studies with the homologous archaeal MCM helicases have revealed that the leading strand must pass through the central pore of the helicase, whereas the lagging strand takes an external path (11–14).Our focus to compare the structure and function of the CMG with Mcm2–7 has been motivated by two considerations. First, understanding the allosteric induction of helicase activity accomplished by the GINS and Cdc45 proteins should inform the mechanism for initiation, the notion here being that the first melting step might be coupled to the formation of the active helicase. Second, the active CMG helicase coordinates the assembly of key proteins for DNA strand synthesis—such as Ctf4 (15), Mcm10 (16), and polε (17)—implying that structural transitions in the Mcm2–7 complex affected by the GINS and Cdc45 may assist in recruitment and function of multiple activities of the replisome. How the Mcm2–7 proteins might influence these interactions or how leading and lagging strand synthesis might be coordinated with unwinding are unknown. Thus, the path of both the leading and lagging strands and the functions of the nonmotor proteins in the complex are important for a more complete picture of the eukaryotic replisome.In previous studies, we have shown that in the CMG, GINS and Cdc45 bridge a gap between the Mcm2 and Mcm5 subunits, and that when a nucleotide is bound, these interactions seal off the interior channel, creating a topologically segregated second channel to the side of the central axis of the Mcm2–7 ring (18). In the apo state, interactions between Mcm2 and Mcm5 change, creating an opening between the subunits that renders the space of the interior channel contiguous with the external side channel. This discontinuity between Mcm2 and Mcm5 was first described biochemically in the yeast Mcm2–7 complex (19) and has been called a “gate,” as opening between the subunits at this position is crucial for loading to substrates in biochemical assays. For the CMG, given that amino acids in Mcm5 and Mcm2 are both required for the helicase activity (4), we reasoned that the requisite ATPase domain interactions between these gate subunits are critical for helicase activity and that activation by GINS/Cdc45 helps facilitate formation of this structure. How the open and closed conformations of the gate and side channel might come into play during helicase activity has remained unexplored.Among the several outstanding mechanistic questions with regard to helicase activity and replication fork organization is the path of the lagging strand and the roles, if any, for Cdc45 and GINS in guiding the lagging strand. From the observed positions of these factors and the nexus of the DNA fork junction in EM reconstructions of the complex (18, 20), we reasoned that neither set of factors is positionally capable of serving as a “wedge” that might facilitate unwinding or that might work behind the helicase in a plow-like capacity. Nevertheless, other studies had shown that both Cdc45 (21–23) and the GINS complex (4, 24) have a weak but measurable DNA binding activity. Insofar as the lagging strand would need to exit the central channel following Mcm2–7 activation and DNA melting, it initially seemed possible that this DNA segment might interact with GINS or Cdc45, which could capture it within the side channel created upon closure of the Mcm2/5 gate (18).To better understand the disposition of DNA segments associated with the CMG, we used protein/nucleic-acid cross-linking methods to define a path for the lagging strand. These studies show that the lagging strand template DNA when bound to the CMG in its translocation mode does not make intimate contact with either Cdc45 or the GINS but that it instead interacts with the MCMs. We refer to this template DNA as “lagging strand” throughout the text. Moreover, we found that in the apo state, the leading strand template DNA cross-links to Cdc45. We refer to this template DNA as “leading strand.” Using site-specific mutations directed to residues in the set of β-hairpin elements that project into the central channel of Mcm2–7 (25) [the so-called “pre-Sensor I” (PS1) motifs], we show that the nucleotide dependency of Cdc45 leading strand cross-links is altered, such that Cdc45–DNA contacts form even when ATP is present. Using a recent, higher resolution EM structure of the CMG bound to a tailed DNA substrate (20), along with homology modeling to prokaryotic orthologs of Cdc45, we identify residues within Cdc45 that ablate cross-linking; surprisingly, these alterations also affect helicase activity. Collectively, these data suggest that the gate between Mcm2 and Mcm5 can open at given points and that on these occasions the leading strand may dissociate from the central channel; in such instances, the side channel formed by GINS and Cdc45 would help prevent CMG dissociation and enable reestablishment of productive translocation. Together, our data underscore a potential role for Cdc45 in maintaining contacts with both the leading and lagging strands when the helicase may be stalled—such as during S-phase stress—and where the open gate conformation may persist, a point emphasized by the homology of Cdc45 to the prokaryotic repair protein RecJ (21, 26).     

A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.     

Origin recognition complex harbors an intrinsic nucleosome remodeling activity

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)₂ tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

DNA replication is a vital life process for all cell types—bacterial, eukaryotic, and archaeal. While there are important differences among the replication proteins of the three domains of life, they mostly function in similar ways. All of them use an origin binding protein that acts with other factors to load two hexameric helicases onto DNA for bidirectional unwinding of the duplex, and thus the ability to simultaneously replicate both strands of the cellular genome (1–3). The eukaryotic origin binding protein is a heterohexamer referred to as the origin recognition complex (ORC) (4). The sequences of the Orc1-6 subunits are conserved from yeast to human, and several of the subunits contain an adenosine triphosphate (ATP)-binding AAA⁺ module as in the Escherichia coli DnaA initiator. Origins in the budding yeast Saccharomyces cerevisiae occur in 100- to 200-bp DNA regions known as autonomously replicating sequences (ARSs) (5–10). However, the existence of ARSs is limited to only some species of budding yeast. Origins of replication with defined DNA sequences are not known at this time to exist in other eukaryotes (1, 2).The special feature of a defined origin sequence in S. cerevisiae has facilitated extensive characterization of the mechanism of DNA replication initiation (1). ORC interacts with Cdc6, Cdt1, and the minichromosome maintenance protein complex (Mcm)2–7 heterohexamer to assemble a Mcm2-7 double hexamer (referred to here as MCM DH) onto DNA in G1 phase (1–3). The loaded MCM DH is the “licensing” factor for replication because it acts as the marker for origin firing in S phase (11). Specifically, the MCM DH is acted upon by several initiation factors to form 2 larger 11-subunit CMG (Cdc45/Mcm2-7/GINS) helicases (12, 13). The two CMG helicases are oriented toward and pass each other to unwind DNA, and recruit the replicative machinery to form bidirectional replication forks (14, 15). ORC and the many other factors required to license an origin and form bidirectional replication forks are conserved in all eukaryotes.The yeast ARS is AT rich, which is not favorable to nucleosome binding (16, 17). Indeed, chromatin immunoprecipitation sequencing (ChIP-seq) studies indicate that many ARSs have a nucleosome-free region (NFR) that expands in G1/S phase (10, 18–20). Presumably the nucleosomes are moved aside to make way for ORC-mediated MCM DH formation at origins in G1 phase, and for CMG formation in S phase. In vitro studies demonstrate that in the presence of saturating nucleosomes, the ARS is functional for replication initiation without need for classic nucleosome remodelers (21), indicating that the expansion of the NFR at an ARS site may be achieved intrinsically by the origin recognition and replication machinery.We have recently reported that ORC binding to nucleosomes facilitates the loading of MCM DHs onto DNA, regardless of the DNA sequence (22). In that study, we observed the loss of the fluorescently labeled histone signal after ORC–nucleosome interaction, but did not investigate further the source and mechanism of this observation as it was not the focus of the study. Considering that ORC binding is the first step of origin licensing and that ORC harbors multiple ATPase sites, here, we explored the possibility that ORC itself may possess an ATP-facilitated nucleosome remodeling activity. Using single-molecule fluorescence microscopy combined with optical trapping, we find that ORC is indeed an ATP-dependent nucleosome remodeler with the ability to eject H2A-H2B dimers. ORC-mediated nucleosome remodeling may represent the inaugural event toward creating a local chromatin environment permissive to replication initiation.     

Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing

In a search for Polo-like kinase 1 (Plk1)-interacting proteins using a yeast two-hybrid system, we have identified histone acetyltransferase binding to the origin recognition complex 1 (Hbo1) as a potential Plk1 target. Here, we show that the interaction between Plk1 and Hbo1 is mitosis-specific and that Plk1 phosphorylates Hbo1 on Ser-57 in vitro and in vivo. During mitosis, Cdk1 phosphorylates Hbo1 on Thr-85/88, creating a docking site for Plk1 to be recruited. Significantly, the overexpression of Hbo1 mutated at the Plk1 phosphorylation site (S57A) leads to cell-cycle arrest in the G₁/S phase, inhibition of chromatin loading of the minichromosome maintenance (Mcm) complex, and a reduced DNA replication rate. Similarly, Hbo1 depletion results in decreased DNA replication and a failure of Mcm complex binding to chromatin, both of which can be partially rescued by the ectopic expression of WT Hbo1 but not Hbo1-S57A. These results suggest that Plk1 phosphorylation of Hbo1 may be required for prereplicative complex (pre-RC) formation and DNA replication licensing.     

A p53-independent damage-sensing mechanism that functions as a checkpoint at the G1/S transition in Chinese hamster ovary cells

In response to a moderate dose of radiation, asynchronous mammalian cell populations rapidly and transiently down-regulate the rate of DNA synthesis to ≈50% of preirradiation values. We show here that only half of the reduction in overall replication rate can be accounted for by direct inhibition of initiation at origins in S-phase cells. The other half results from the operation of a newly defined cell cycle checkpoint that functions at the G₁/S transition. This checkpoint senses damage incurred at any time during the last 2 hr of G₁ and effectively prevents entry into the S period. The G₁/S and S-phase checkpoints are both p53-independent and, unlike the p53-mediated G₁ checkpoint, respond rapidly to radiation, suggesting that they may represent major damage-sensing mechanisms connecting the replication machinery with DNA repair pathways.