CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.     

Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control

Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae. CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.     

A role for the nuclear envelope in controlling DNA replication within the cell cycle

In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.     

The DNA replication checkpoint response stabilizes stalled replication forks

In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase. Active Rad53 slows DNA synthesis when DNA is damaged and prevents firing of late origins of replication. Further, rad53 mutants are unable to recover from a replication block. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.     

CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast

In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref. 2) is an essential CDK substrate, but its phospho-mimetic form (Sld2-11D) alone neither affects cell growth nor promotes DNA replication in the absence of CDK activity, suggesting that other essential CDK substrates promote DNA replication. Here we show that both an allele of CDC45 (JET1) and high-copy DPB11, in combination with Sld2-11D, separately confer CDK-independent DNA replication. Although Cdc45 is not an essential CDK substrate, CDK-dependent phosphorylation of Sld3, which associates with Cdc45 (ref. 5), is essential and generates a binding site for Dpb11. Both the JET1 mutation and high-copy DPB11 by-pass the requirement for Sld3 phosphorylation in DNA replication. Because phosphorylated Sld2 binds to the carboxy-terminal pair of BRCT domains in Dpb11 (ref. 4), we propose that Dpb11 connects phosphorylated Sld2 and Sld3 to facilitate interactions between replication proteins, such as Cdc45 and GINS. Our results demonstrate that CDKs regulate interactions between BRCT-domain-containing replication proteins and other phosphorylated proteins for the initiation of chromosomal DNA replication; similar regulation may take place in higher eukaryotes.     

Replication fork movement sets chromatin loop size and origin choice in mammalian cells

Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented, but much less is known about the mechanisms controlling the spacing of initiation events(2,3), namely the completion of DNA replication. Here we show that origin use in Chinese hamster cells depends on both the movement of the replication forks and the organization of chromatin loops. We found that slowing the replication speed triggers the recruitment of latent origins within minutes, allowing the completion of S phase in a timely fashion. When slowly replicating cells are shifted to conditions of fast fork progression, although the decrease in the overall number of active origins occurs within 2 h, the cells still have to go through a complete cell cycle before the efficiency specific to each origin is restored. We observed a strict correlation between replication speed during a given S phase and the size of chromatin loops in the next G1 phase. Furthermore, we found that origins located at or near sites of anchorage of chromatin loops in G1 are activated preferentially in the following S phase. These data suggest a mechanism of origin programming in which replication speed determines the spacing of anchorage regions of chromatin loops, that, in turn, controls the choice of initiation sites.     

The Cdt1 protein is required to license DNA for replication in fission yeast

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 cycle. 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 chromatin. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated 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.     

Identification of a novel resident centrosomal protein

One human autoimmune serum was identified to react with centrosomes by immunofluorescence. We applied the affinity purification of membrane-bound antibody technique and demonstrated that the antibodies present in this antiserum reacted with a 31/29 ku centrosomal antigen. Immunofluorescence showed that this antigen is located at centrosome in a cell-cycle independent manner, and thereby it belongs to the family of centrosomal residents. We then uti- lized this autoimmune serum and antibodies against centrin and gamma-tubulin to investigate changes of centrosome cycle kinetics during premature chromosome condensation (PCC) artificially induced in V79-8 cells. We show here that centrosomal proteins continue to express when cells are synchronized at G1/S boundary and S phase by Hydroxyurea (HU). During this time, the addition of caffeine causes cells with unreplicated genome to go into mitosis, and induces the separation of the replicated centrosomes. These results suggest that the coordination of DNA synthesis and centrosome replication in the normal cell cycle can be uncoupled. Cells ensure that centrosome duplicates once, and only once during each DNA synthesis cycle through the tight and subtle coordination of cell cycle engine molecules, and thereby the assembly of bipolar spindle and the accurate transmission of genetic information.     

Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

Break-induced replication and telomerase-independent telomere maintenance require Pol32

Break-induced replication (BIR) is an efficient homologous recombination process to initiate DNA replication when only one end of a chromosome double-strand break shares homology with a template. BIR is thought to re-establish replication at stalled and broken replication forks and to act at eroding telomeres in cells that lack telomerase in pathways known as 'alternative lengthening of telomeres' (reviewed in refs 2, 6). Here we show that, in haploid budding yeast, Rad51-dependent BIR induced by HO endonuclease requires the lagging strand DNA Polalpha-primase complex as well as Poldelta to initiate new DNA synthesis. Polepsilon is not required for the initial primer extension step of BIR but is required to complete 30 kb of new DNA synthesis. Initiation of BIR also requires the nonessential DNA Poldelta subunit Pol32 primarily through its interaction with another Poldelta subunit, Pol31. HO-induced gene conversion, in which both ends of a double-strand break engage in homologous recombination, does not require Pol32. Pol32 is also required for the recovery of both Rad51-dependent and Rad51-independent survivors in yeast strains lacking telomerase. These results strongly suggest that both types of telomere maintenance pathways occur by recombination-dependent DNA replication. Thus Pol32, dispensable for replication and for gene conversion, is uniquely required for BIR; this finding provides an opening into understanding how DNA replication re-start mechanisms operate in eukaryotes. We also note that Pol32 homologues have been identified both in fission yeast and in metazoans where telomerase-independent survivors with alternative telomere maintenance have also been identified.     

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.     

Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions

DNA lesions can often block DNA replication, so cells possess specialized low-fidelity, and often error-prone, DNA polymerases that can bypass such lesions and promote replication of damaged DNA. The Saccharomyces cerevisiae RAD30 and human hRAD30A encode Pol eta, which bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Here we show that a related human gene, hRAD30B, encodes the DNA polymerase Pol iota, which misincorporates deoxynucleotides at a high rate. To bypass damage, Pol iota specifically incorporates deoxynucleotides opposite highly distorting or non-instructional DNA lesions. This action is combined with that of DNA polymerase Pol zeta, which is essential for damage-induced mutagenesis, to complete the lesion bypass. Pol zeta is very inefficient in inserting deoxynucleotides opposite DNA lesions, but readily extends from such deoxynucleotides once they have been inserted. Thus, in a new model for mutagenic bypass of DNA lesions in eukaryotes, the two DNA polymerases act sequentially: Pol iota incorporates deoxynucleotides opposite DNA lesions, and Pol zeta functions as a mispair extender.     

ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.

A multiprotein complex that specifically recognizes cellular origins of DNA replication has been identified and purified from the yeast Saccharomyces cerevisiae. We observe a strong correlation between origin function and origin recognition by this activity. Interestingly, specific DNA binding by the origin recognition complex is dependent upon the addition of ATP. We propose that the origin recognition complex acts as the initiator protein for S. cerevisiae origins of DNA replication.