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.     

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.     

Competence to replicate in the unfertilized egg is conferred by Cdc6 during meiotic maturation

Meiotic maturation, the final step of oogenesis, is a crucial stage of development in which an immature oocyte becomes a fertilizable egg. In Xenopus, the ability to replicate DNA is acquired during maturation at breakdown of the nuclear envelope by translation of a DNA synthesis inducer that is not present in the oocyte. Here we identify Cdc6, which is essential for recruiting the minichromosome maintenance (MCM) helicase to the pre-replication complex, as this inducer of DNA synthesis. We show that maternal cdc6 mRNA but not protein is stored in the oocyte. Cdc6 protein is synthesized during maturation, but this process can be blocked by degrading the maternal cdc6 mRNA by oligonucleotide antisense injections or by translation inhibition. Rescue experiments using recombinant Cdc6 protein show that Cdc6 is the only missing replication factor whose translation is necessary and sufficient to confer DNA replication competence to the egg before fertilization. The licence to replicate is given by Cdc6 at the end of meiosis I, but the cytostatic factor (CSF) pathway, which maintains large amounts of active Cdc2/Cyclin B2, prevents the entry into S phase until fertilization.     

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 RCAF complex mediates chromatin assembly during DNA replication and repair

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.     

The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.     

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.     

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.     

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.     

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.     

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.     

Structural basis for inhibition of the replication licensing factor Cdt1 by geminin

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. Here we describe the crystal structure of the mouse geminin-Cdt1 complex using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance.     

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.