Abstract: Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

Abstract: DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity. These studies also reveal polymerase interactions with the DNA minor groove at and upstream of the active site that influence nucleotide selectivity, the efficiency of exonucleolytic proofreading, and the rate of forming errors via strand misalignments. We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.

Abstract: In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.

Abstract: DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.

Abstract: To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle1. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin2,3,4,5,6,7,8,9,10. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis12. Other factors acting in this control have been postulated13 and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.

Abstract: In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G1-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of ARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase a onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase « only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.

Abstract: The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. Here we have expressed and purified the M. thermoautotrophicum MCM protein. The purified protein forms a complex that has a molecular mass of ≈850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3′ to 5′ helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase. It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea. The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.

Abstract: The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content. Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophage-like, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A. One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript. In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus. Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects. Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.

Abstract: Using methods that conserve nuclear architecture, we have reanalyzed the spatial organization of the initiation of mammalian DNA synthesis. Contrary to the commonly held view that replication begins at hundreds of dispersed nuclear sites, primary fibroblasts initiate synthesis in a limited number of foci that contain replication proteins, surround the nucleolus, and overlap with previously identified internal lamin A/C structures. These foci are established in early G(1)-phase and also contain members of the retinoblastoma protein family. Later, in S-phase, DNA replication sites distribute to regions located throughout the nucleus. As this progression occurs, association with the lamin structure and pRB family members is lost. A similar temporal progression is found in all the primary cells we have examined but not in most established cell lines, indicating that the immortalization process modifies spatial control of DNA replication. These findings indicate that in normal mammalian cells, the onset of DNA synthesis is coordinately regulated at a small number of previously unrecognized perinucleolar sites that are selected in early G(1)-phase.

Abstract: CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G1. A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G1 and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC–CDH1-dependent proteolysis of CDC6 in early G1 and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G1.

Abstract: We have examined the cellular function of Sgs1p, a nonessential yeast DNA helicase, homologs of which are implicated in two highly debilitating hereditary human diseases (Werner's and Bloom's syndromes). We show that Sgs1p is an integral component of the S-phase checkpoint response in yeast, which arrests cells due to DNA damage or blocked fork progression during DNA replication. DNA pole and Sgs1p are found in the same epistasis group and act upstream of Rad53p to signal cell cycle arrest when DNA replication is perturbed. Sgs1p is tightly regulated through the cell cycle, accumulates in S phase and colocalizes with Rad53p in S-phase-specific foci, even in the absence of fork arrest. The association of Rad53p with a chromatin subfraction is Sgs1p dependent, suggesting an important role for the helicase in the signal-transducing pathway that monitors replication fork progression.

Abstract: Eukaryotic chromosomal DNA replicates exactly once per cell cycle, in the S phase. In Saccharomyces cerevisiae, chromosomal DNA replication is initiated at a restricted region known as the autonomously replicating sequence (ARS) (reviewed in references 10 and 43). An origin recognition complex (ORC), comprising six subunits, is bound to each ARS throughout the cell cycle (6, 14). At the end of mitosis, six Mcm family proteins (Mcm2 to -7) are also loaded with the ORC onto the ARS as components of the prereplicative complex (pre-RC) (14). The Mcm proteins have a conserved amino acid sequence and form large complexes (11, 29, 48). Loading of these proteins requires both the ORC and Cdc6 (1, 15, 46). At the onset of S phase, Cdk and Cdc7 protein kinases facilitate loading of the single-stranded DNA binding protein, RPA, onto the ARS (47). Then, the three DNA polymerases (Polα, -δ, and -ɛ), essential for chromosomal DNA replication (reviewed in reference 45), are recruited to the ARS region to initiate DNA synthesis (1, 2, 47). Association of Polα and Polɛ with the ARS region is dependent on Cdc45 (2), which associates with the ARS in the G1 and S phases of the cell cycle (1, 2) and interacts with the Mcm proteins (12, 21, 22, 24, 32, 52). All the replication proteins described above are well conserved from yeasts through humans. The Cdc45 protein of Xenopus laevis is also required for association of Polα with chromatin DNA (31), as observed in yeast. Moreover, Cdc45 and Polα form a complex in Xenopus egg extracts (31). In in vitro simian virus 40 (SV40) DNA replication, T antigen unwinds the replication origin and RPA binds the unwound single-stranded DNA. DNA primase, tightly associated with Polα, then synthesizes an RNA primer. This RNA primer is used by Polα to synthesize a short DNA strand, followed by elongation of the DNA strand by Polδ and/or Polɛ, using the short DNA fragment as a primer (43). Although the cellular counterpart of the SV40 T antigen has not been identified, it is believed that similar reactions take place during chromosomal DNA replication. DNA replication in eukaryotic cells initiates from multiple replication origins that fire throughout the S phase of the cell cycle; some origins fire early, others fire late (17, 25). In S. cerevisiae, hydroxyurea (HU), which inhibits ribonucleotide reductase and consequently inhibits DNA synthesis, blocks the firing of late origins. In rad53 and mec1 mutants (defective in cell cycle checkpoints), however, late-origin firing is not blocked by HU (40). Methyl methane sulfonate similarly inhibits late-origin firing, and this inhibition is not observed in rad53 or orc2 mutants (42). From these observations, it is proposed that the regulation of late-origin firing is important for the S-phase checkpoint (13, 40, 42). The DPB11 gene was isolated as a multicopy suppressor of mutations in the POL2 and DPB2 genes, which encode the catalytic and second-largest subunits of Polɛ, respectively (5). The amino acid sequence of Dpb11 is similar to the sequence of the Cut5 (also known as Rad4) protein of Schizosaccharomyces pombe. This protein is required for the onset of the S-phase and cell cycle checkpoint in S. pombe (30, 37–39, 49). Both Dpb11 and Cut5 have four copies of the BRCA1 C-terminus (BRCT) domain, which is thought to be an interaction domain between proteins (7, 9, 51). In thermosensitive dpb11-1 mutants, S-phase progression is delayed when the temperature is shifted up, followed by cell division with unequal chromosome segregation. In the presence of HU, dpb11-1 cells also have an elongated spindle, indicating that mitosis has started without the completion of DNA replication. Furthermore, there is a strong genetic interaction between Dpb11 and Polɛ; high-copy DPB11 suppresses the growth defects of pol2-11 and dpb2-1, and no combination of dpb11-1 with one of pol2-11, pol2-18, and dpb2-1 is obtained. This suggests that Dpb11 interacts with Polɛ and is required for DNA replication and the S-phase checkpoint (5). To gain a broader understanding of the function of Dpb11, we tried to identify the factors that interact with Dpb11 by isolating synthetic lethal mutations with dpb11-1 (sld). So far, we have isolated five SLD genes. SLD1 is identical to the DPB3 gene that encodes the third-largest subunit of Polɛ (4), and SLD4 is identical to CDC45 (22). The SLD2 gene encodes a 52-kDa protein that forms a complex with Dpb11 that is essential for DNA replication. From this analysis, we suggested that a Dpb11-Sld2 complex is required for one of the steps close to the initiation of DNA replication (27). The SLD2 gene was independently isolated as the DRC1 (DNA replication and checkpoint 1) gene, and the drc1-8 mutant was found to be defective in the S-phase checkpoint (50). To further elucidate the function of Dpb11, we analyzed the association between Dpb11, DNA polymerases, and chromatin DNA, using a chromatin immunoprecipitation (CHIP) assay (44). In the S phase of the cell cycle, Dpb11 and Polɛ simultaneously associated with DNA fragments containing an ARS, and their association was mutually dependent. We also detected a complex of Dpb11 and Pol2 that was most abundant during the S phase. Moreover, Dpb11 was required for blockage of late-origin firing by HU. Therefore, we suggest that Dpb11 is required for DNA polymerases to associate with the ARS and for blockage of late-origin firing.

Abstract: The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.

Abstract: The initiation of DNA replication forks occurs at replication origins distributed along chromosomes and must be strictly controlled to ensure that the DNA is replicated once and once only in each cell cycle (for review, see Diffley 1996; Stillman 1996; Donaldson and Blow 1999). The process of building an active replication origin can be divided into two phases. The first phase, occurring in late mitosis and early G1, comprises the sequential assembly onto replication origins of the origin recognition complex (ORC), the Cdc6 protein, and the RLF–M complex of Mcm2–7 proteins (also known as MCM/P1 proteins), which results in them becoming “licensed” for DNA replication in the subsequent S phase. The second phase involves the action of the Cdc7 and cyclin-dependent kinases (CDKs) on each origin to load the Cdc45 protein and to induce the initiation of a pair of replication forks. It is of considerable current interest to understand in more detail the sequence of events leading to the initiation of replication. In this paper we have concentrated on defining the precise stage in the process when the Cdc7 protein acts, using the biochemically tractable Xenopus cell-free DNA replication system. Replication origins appear to be defined by binding the ORC (Bell and Stillman 1992). In yeast, ORC is bound to origins throughout the cell cycle (Diffley and Cocker 1992; Diffley et al. 1994), whereas in higher eukaryotes, ORC is probably displaced from the DNA during mitosis (Coleman et al. 1996; Romanowski et al. 1996; Rowles et al. 1999). During late mitosis and early G1, Cdc6 is then assembled onto ORC-containing DNA (Coleman et al. 1996). Chromatin containing ORC and Cdc6 can then be licensed by loading the RLF-M complex of MCM/P1 proteins, a reaction also requiring the RLF-B component of the replication licensing system (Chong et al. 1995; Kubota et al. 1995, 1997; Thommes et al. 1997; Tada et al. 1999; Prokhorova and Blow 2000). The complex of ORC, Cdc6, and MCM/P1 proteins appears to be responsible for the footprint of the prereplicative complex (pre-RC) observed in Saccharomyces cerevisiae on replication origins in late mitosis and early G1 (Diffley et al. 1994). Once licensing has occurred, ORC and Cdc6 become more loosely bound to DNA and have fulfilled their essential function in DNA replication (Hua and Newport 1998; Rowles et al. 1999). For a licensed origin to initiate replication, two S phase-promoting protein kinases are then required: an S phase-promoting CDK and the Cdc7/Dbf4 protein kinase. Cdc7 is a serine threonine kinase conserved from yeast to humans that is required for the initiation of DNA replication (Hollingsworth et al. 1992; Jackson et al. 1993; Masai et al. 1995; Jiang and Hunter 1997; Sato et al. 1997; Hess et al. 1998). Although Cdc7 protein levels are approximately constant throughout the cell cycle, Cdc7 kinase activity peaks at the G1/S transition (Jackson et al. 1993; Yoon et al. 1993). This regulation is achieved in part by association with a regulatory subunit termed Dbf4 (Brown and Kelly 1999; Cheng et al. 1999; Jiang et al. 1999; Oshiro et al. 1999; Takeda et al. 1999). Instead of acting as a general initiator of S phase, Cdc7 is probably required to promote initiation at individual origins because, as well as being required for progression into early S phase, it is also required late in S phase to promote initiation at late-firing origins (Bousset and Diffley 1998; Donaldson et al. 1998a). Several lines of evidence suggest that the MCM/P1 proteins are the physiological substrate of Cdc7/Dbf4. In yeast, a mutant allele of Mcm5 (mcm5–bob1) can suppress a complete loss of Cdc7 or Dbf4 (Hardy et al. 1997). Conversely, a mutant of Dbf4 (dbf4-6) was isolated as an allele-specific suppressor of Mcm2 (Lei et al. 1997). Cdc7 and Dbf4 interact physically with MCM/P1 proteins (Lei et al. 1997; Roberts et al. 1999), and a number of reports have identified MCM/P1 proteins as excellent substrates of Cdc7/Dbf4 kinase in vitro (Lei et al. 1997; Sato et al. 1997). Moreover, certain phosphorylations of the MCM/P1 proteins (most notably of Mcm2) in vivo depend on Cdc7 (Lei et al. 1997; Jiang et al. 1999). The ability of Cdc7 to phosphorylate the MCM/P1 proteins may be aided by its recruitment to replication origins because, in yeast, both Dbf4 and Cdc7 have been shown to interact with chromatin (Pasero et al. 1999; Weinreich and Stillman 1999), the association being dependent on ORC but not Cdc6 (Pasero et al. 1999). This interaction may be mediated by Dbf4, because a one-hybrid screen identified a domain of Dbf4 distinct from the Cdc7 interaction domain that recruited Dbf4 to replication origins (Dowell et al. 1994). Cdc7/Dbf4 could be a target of checkpoint kinases, because when yeast cells were treated with hydroxyurea to block progression through S phase, Dbf4 dissociated from the chromatin (Pasero et al. 1999). This treatment also induced the appearance of a phosphorylated form of Dbf4, which was dependent on the checkpoint kinase Rad53 (Brown and Kelly 1999; Cheng et al. 1999; Takeda et al. 1999; Weinreich and Stillman 1999). CDKs are also required for the initiation of licensed replication origins. CDK activity leads to the assembly of the essential initiation protein Cdc45 onto origins, creating a “preinitiation complex” (Zou and Stillman 1998). Cdc45 interacts genetically with different components of the pre-RC including MCM/P1 proteins and ORC (Hopwood and Dalton 1996; Owens et al. 1997; Zou et al. 1997), and the association of Cdc45 with chromatin requires Cdc6 and Mcm2 (Zou and Stillman 1998). However, the binding of yeast Cdc45 to chromatin is independent of Cdc7 function, suggesting that CDKs and Cdc7 may act on parallel pathways to initiate replication (Zou and Stillman 1998). CDKs play an important role in executing the temporal program of origin activation during the course of S phase (Donaldson et al. 1998b). Like Cdc7, CDKs are probably required to promote initiation at individual origins, because the CDK-dependent loading of Cdc45 onto late-firing origins only occurs late in S phase (Aparicio et al. 1999). In the present report, we have used cell-free extracts of Xenopus eggs to determine the precise stage in the process of origin activation at which Xenopus (X) Cdc7 acts. We show that XCdc7 binds to chromatin during G1 and S phase and that both the chromatin binding and the essential DNA replication function of XCdc7 are dependent on licensing but do not require the presence of XORC or XCdc6. We show that XCdc7 is required for the subsequent loading of XCdc45 onto chromatin by CDKs. Finally we show that XCdc7-dependent phosphorylation of XMcm2 and the essential function of XCdc7 can be performed in the absence of CDK activity. These results provide a simple model for the function of Cdc7 in the Xenopus system that appears to differ from that occurring in yeast.

Abstract: Here we show that exposure of aphidicolin-arrested Chinese hamster ovary (CHO) cells to the protein-kinase inhibitors 2-aminopurine or caffeine results in initiation of replication at successively later-replicating chromosomal domains, loss of the capacity to synthesize DNA at earlier-replicating sites, release of Mcm2 proteins from chromatin, and redistribution of PCNA and RPA from early- to late-replicating domains in the absence of detectable elongation of replication forks. These results provide evidence that, under conditions of replicational stress, checkpoint controls not only prevent further initiation but may also be required to actively maintain the integrity of stalled replication complexes.

Abstract: Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes in HeLa cells were modified by the expression of different cellular or viral membrane-binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting PV multiplication. Our data suggest that a functional replication complex is formed in cis, in a coupled process involving viral translation, membrane modification and vesicle budding, and viral RNA synthesis.

Abstract: Background In vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently, a family of methyl-CpG-binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive. Results It was shown that MBD2 and MBD3 form homo- and hetero-dimers (or multimers) in vitro and in vivo. Significantly, the MBD2-MBD3 complex showed an affinity to hemi-methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co-localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a co-immunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle. Conclusions The results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2-MBD3 complex recognizes hemi-methylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.

Abstract: Great insight into the molecular details of cell cycle regulation has been obtained in the past decade. However, most of the progress has been in defining the regulation of the family of cyclin-dependent kinases (CDKs). Recent studies of a myriad of eukaryotic organisms have defined both the regulation and substrates of Cdc7p kinase, which forms a CDK-cyclin-like complex with Dbf4p, is necessary for the initiation of DNA replication and has been conserved in evolution. This kinase is also required for the induction of mutations after DNA damage and for commitment to recombination in the meiotic cell cycle. However, less is known about the role of the kinase in these processes. In a manner similar to CDKs, Cdc7p is activated by a regulatory subunit, Dbf4, the levels of which fluctuate during the cell cycle. One or more subunits of the conserved MCM helicase complex at chromosomal origins of DNA replication are substrates for the kinase during S phase. Phosphorylation of the MCM complex by Cdc7p-Dbf4p might activate DNA replication by unwinding DNA. Therefore, activation of Cdc7p is required for DNA replication. Given that Cdc7p-Dbf4 kinase is overexpressed in many neoplastic cells and tumors, it might be an important early biomarker during cancer progression.

Abstract: In Escherichia coli cells, the origin of chromosomal replication is temporarily inactivated after initiation has occurred. Origin sequestration is the first line of defence against over-initiation, providing a time window during which the initiation potential can be reduced by: (i) titration of DnaA proteins to newly replicated chromosomal elements; (ii) regulation of the activity of the DnaA initiator protein; and (iii) sequestration of the dnaA gene promoter. This review represents the first attempt to consider together older and more recent data on such inactivation mechanisms in order to analyze their contributions to the overall tight replication control observed in vivo. All cells have developed mechanisms for origin inactivation, but those of other bacteria and eukaryotic cells are clearly distinct from those of E. coli. Possible differences and similarities are discussed.

Abstract: by a regulatory subunit, Dbf4, the levels of which fluctuate during the cell cycle. One or more subunits of the conserved MCM helicase complex at chromosomal origins of DNA replication are substrates for the kinase during S phase. Phosphorylation of the MCM complex by Cdc7p-Dbf4p might activate DNA replication by unwinding DNA. Therefore, activation of Cdc7p is required for DNA replication. Given that Cdc7p-Dbf4 kinase is overexpressed in many neoplastic cells and tumors, it might be an important early biomarker during cancer progression. SUMMARY

Abstract: Background In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood. Results Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase α and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase ɛ onto chromatin required Cdc45 but did not require DNA polymerase α. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase α on chromatin. Conclusions These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA.