Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion.     

Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts

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 alpha and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase epsilon onto chromatin required Cdc45 but did not require DNA polymerase alpha. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase alpha 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.     

Replisome progression complex links DNA replication to sister chromatid cohesion in Xenopus egg extracts

Cohesin-mediated sister chromatid cohesion is established during the S-phase, and recent studies demonstrate that a cohesin protein ring concatenates sister DNA molecules. However, little is known about how DNA replication is linked to the establishment of sister chromatid cohesion. Here, we used Xenopus egg extracts to show that AND-1 and Tim1–Tipin, homologues of Saccharomyces cerevisiae Ctf4 and Tof1–Csm3, respectively, are associated with the replisome and are required for proper establishment of the cohesion observed in the M-phase extracts. Immunodepletion of both AND-1 and Tim1–Tipin from the extracts leads to aberrant sister chromatid cohesion, which is similarly induced by the depletion of cohesin. These results demonstrate that AND-1 and Tim1–Tipin are key factors linking DNA replication and establishment of sister chromatid cohesion. On the basis of the physical interactions between AND-1 and DNA polymerases, we discuss a model to describe how replisome progression complex establishes sister chromatid cohesion.     

Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts

In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. Here, we show that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase alpha, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. Our results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase.     

Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts

DNA polymerases delta and epsilon (Poldelta and Polepsilon) are widely thought to be the major DNA polymerases that function in elongation during DNA replication in eukaryotic cells. However, the precise roles of these polymerases are still unclear. Here we comparatively analysed DNA replication in Xenopus egg extracts in which Poldelta or Polepsilon was immunodepleted. Depletion of either polymerase resulted in a significant decrease in DNA synthesis and accumulation of short nascent DNA products, indicating an elongation defect. Moreover, Poldelta depletion caused a more severe defect in elongation, as shown by sustained accumulation of both short nascent DNA products and single-stranded DNA gaps, and also by elevated chromatin binding of replication proteins that function more frequently during lagging strand synthesis. Therefore, our data strongly suggest the possibilities that Poldelta is essential for lagging strand synthesis and that this function of Poldelta cannot be substituted for by Polepsilon.     

Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts

The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.     

A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication

We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.     

Intrinsic nuclear import activity of geminin is essential to prevent re-initiation of DNA replication in Xenopus eggs

Prior to S phase, eukaryotic chromosomes are licensed for initiation of DNA replication, and re-licensing is prohibited after S phase has started until late mitosis, thus ensuring that genomic DNA is duplicated precisely once in each cell cycle. Here, we report that over-expression of Cdt1, an essential licensing protein, induced re-replication in Xenopus egg extracts. Geminin, a metazoan-specific inhibitor of Cdt1, was critical for preventing re-replication induced by Cdt1. Re-replication induced by the addition of recombinant Cdt1 and/or by the depletion of geminin from extracts was enhanced by a proteasome inhibitor, which suppressed the degradation of Cdt1 in the extracts. Furthermore, a nuclear localization sequence identified in Xenopus geminin had a significant role in the suppression of re-replication induced by Cdt1. These results suggest that nuclear accumulation of geminin plays a dominant role in the licensing system of Xenopus eggs.     

Papillomavirus DNA replication — From initiation to genomic instability

Papillomaviruses establish their productive life cycle in stratified epithelium or mucosa, where the undifferentiated proliferating keratinocytes are the initial targets for the productive viral infection. Papillomaviruses have evolved mechanisms to adapt to the normal cellular growth control pathways and to adjust their DNA replication and maintenance cycle to contend with the cellular differentiation. We provide overview of the papillomavirus DNA replication in the differentiating epithelium and describe the molecular interactions important for viral DNA replication on all steps of the viral life cycle.     

Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication

Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. Here, we report that in Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus.     

Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication

The MCM2-7 helicase complex is loaded on DNA replication origins during the G1 phase of the cell cycle to license the origins for replication in S phase. How the initiator primase-polymerase complex, DNA polymerase alpha (pol alpha), is brought to the origins is still unclear. We show that And-1/Ctf4 (Chromosome transmission fidelity 4) interacts with Mcm10, which associates with MCM2-7, and with the p180 subunit of DNA pol alpha. And-1 is essential for DNA synthesis and the stability of p180 in mammalian cells. In Xenopus egg extracts And-1 is loaded on the chromatin after Mcm10, concurrently with DNA pol alpha, and is required for efficient DNA synthesis. Mcm10 is required for chromatin loading of And-1 and an antibody that disrupts the Mcm10-And-1 interaction interferes with the loading of And-1 and of pol alpha, inhibiting DNA synthesis. And-1/Ctf4 is therefore a new replication initiation factor that brings together the MCM2-7 helicase and the DNA pol alpha-primase complex, analogous to the linker between helicase and primase or helicase and polymerase that is seen in the bacterial replication machinery. The discovery also adds to the connection between replication initiation and sister chromatid cohesion.     

Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53

When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis. In budding yeast, this pathway includes the kinases Mec1 and Rad53. Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks. Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication fork. Blocking initiation of DNA replication blocks Mrc1 loading onto origins, providing an explanation for why so many mutants in DNA replication show checkpoint defects. In the presence of replication blocks, we find that Mec1 is recruited to regions of stalled replication, where it encounters and presumably phosphorylates Mrc1. Mutation of the canonical Mec1 phosphorylation sites on Mrc1 prevents Mrc1 phosphorylation and blocks Rad53 activation, but does not alter Mrc1's role in DNA replication. Our results suggest a model whereby in response to DNA replication interference, the Mec1 kinase is recruited to sites of replication blocks and phosphorylates a component of the DNA replication complex, Mrc1, thereby setting up a solid-state Rad53 activation platform to initiate the checkpoint response.     

Adenovirus DNA synthesis is coupled to virus assembly

The relationship between viral DNA synthesis and virion assembly was studied in adenovirus type 2 infected HEp2 cells. When cells were infected at the restrictive temperature with ts3, an assembly-negative mutant which permits normal viral DNA and protein synthesis, labeled and shifted to the permissive temperature, only de novo synthesized nonradioactive viral DNA was encapsidated. This suggested that only concurrently synthesized DNA is encapsidated. Blocking protein or DNA synthesis with cycloheximide or hydroxyurea after the temperature shift inhibited virus assembly. Therefore efficient virus assembly requires both concurrent protein and DNA synthesis. When DNA synthesis was arrested by shifting ts125 infected cells to the restrictive temperature, protein synthesis continued but assembly was completely blocked. Sucrose gradient sedimentation analysis of nuclear extracts of wt and ts3 infected cells provided evidence in support of a physical coupling between replication complexes and virus assembly complexes. Further evidence of coupling was also shown by preferential pulse labeling of the molecular right end of the genome isolated from reversibly cross-linked assembly intermediate particles. While DNA replication is not dependent on concurrent virion assembly, at least some significant proportion of replication complexes appear to be coupled to and are prerequisite for virion assembly.     

DNA methyltransferase 1 is essential for initiation of the colon cancers

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1^−/−). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR⁺) and CD44-positive and CD24-positive (CD44⁺CD24⁺) cell rates were lower in DNMT1^−/− cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1^−/− cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1^−/− cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.     

The protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi 29 is involved in the initiation of DNA replication

Bacillus subtilis was infected at 30° with θ29 mutants ts in each of four cistrons involved in phage DNA synthesis and, after a short pulse, the infected bacteria were shifted up to 42°C. Analysis of the labeled DNA, at different times after the shift-up, by alkaline sucrose gradient centrifugation showed that unit-length DNA was formed after infection with mutant ts3(132), suggesting a role of protein p3 in the initiation of replication. A similar result was obtained after ts2(98) infection. On the contrary, mutant ts5(17) did not produce unit-length phage DNA even at very late times after the shift-up, consistent with the possibility that this mutation affects an elongation (or maturation) process. Mutant ts6(1360) gave rise to unit-length DNA late after the shift-up suggesting that the mutation might affect an elongation or maturation process but that either it is leaky or that its function may be slowly replaced by a bacterial function. Infection of B. subtilis, under restrictive conditions, with sus mutants in cistron 3 produced unit-length θ29 DNA, both in neutral and alkaline sucrose gradients, suggesting that the parental protein itself present in the sus3 mutants is able to initiate the viral replication.     

Chromosomal location of the DnaA‐reactivating sequence DARS2 is important to regulate timely initiation of DNA replication in Escherichia coli

In Escherichia coli, the initiator protein ATP‐DnaA promotes initiation of chromosome replication in a timely manner. After initiation, DnaA‐bound ATP is hydrolyzed to yield ADP‐DnaA, which is inactive in initiation. DnaA‐reactivating sequences (DARS1 and DARS2) on the chromosome have predominant roles in catalysis of nucleotide exchange, producing ATP‐DnaA from ADP‐DnaA, which is prerequisite for timely initiation. Both DARS sequences have a core region containing a cluster of three DnaA‐binding sites. DARS2 is more effective in vivo than DARS1, and timely activation of DARS2 depends on binding of two nucleoid‐associated proteins, IHF and Fis. DARS2 is located centrally between the chromosomal replication origin oriC and the terminus region terC. We constructed mutants in which DARS2 was translocated to several chromosomal loci, including sites proximal to oriC and to terC. Replication initiation was inhibited in cells in which DARS2 was translocated to terC‐proximal sites when the cells were grown at 42 °C, although overall binding efficiency of IHF and Fis to the translocated DARS2 was not affected. Inhibition was largely sustained even in cells lacking MatP, a DNA‐binding protein responsible for terC‐specific subchromosomal structure. These results suggest that functional regulation of DARS2 is correlated with its chromosomal location under certain conditions.     

A stem-loop structure, sequence non-specific, at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. In this work, a head-to-tail tandem construct of porcine circovirus capable of generating unit-length genomic DNA in Escherichia coli was employed to examine the role of the stem-loop structure with respect to the RCR initiation and termination process. The advantage of using a head-to-tail tandem construct is that the initiation and termination sites for generation of the unit-length viral genomes are physically separated, which allows independent examination of the initiation/termination processes. Nucleotide substitution mutational analysis showed that a pair of inverted repeats capable of forming a stem-loop structure was essential for termination, but not for initiation. The results also demonstrated that it is the stem-loop configuration, not nucleotide sequence specificity, that is critical for terminating RCR DNA replication.     

Characterization of DNA synthesis and chloroplast DNA replication initiation in a Petunia hybrida chloroplast lysate system

Summary An in vitro chloroplast DNA synthesizing lysate system prepared from purified chloroplasts of Petunia hybrida leaves has been developed. Both co-isolated endogeneous chloroplast (cp)DNA and externally added DNA can be used as DNA templates in the system. The system contains a -like DNA polymerase as determined by using DNA polymerase-specific inhibitors and synthetic templates. The molecular weight of this enzyme is about 85 kd. Part of the DNA synthesizing activity is repair synthesis. When a chimaeric plasmid containing a fragment with a potential cpDNA replication origin is used as a template (pPCY62), specific initiation of DNA synthesis is observed on this fragment which strongly suggests that the in vitro chloroplast lysate system is also capable of replication initiation.